ポスター
B. 発生・再生と可塑性
B. Development, Regeneration and Plasticity |
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| 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-105
小頭症、小人症の原因遺伝子CEP152変異マウスは中心体複製障害と細胞死依存的な矮小発育症状を呈する
CEP152-deficiency promotes impaired centrosome duplication- and cell death-dependent dwarfism in mice
*浜田 奈々子(1)、西條 琢磨(1)、西川 将司(1)、上原 朋子(2)、武内 俊樹(2)、小崎 健次郎(2)、水野 誠司(3)、永田 浩一(1)
1. 愛知県医療療育総合センター発達障害研究所、2. 慶応義塾大学医学部臨床遺伝学センター、3. 愛知県医療療育総合センター中央病院
*Nanako Hamada(1), Takuma Nishijo(1), Masashi Nishikawa(1), Tomoko Uehara(2), Toshiki Takenouchi(2), Kenjiro Kosaki(2), Seiji Mizuno(3), Koh-ichi Nagata(1)
1. Aichi Developmental Disability Center, Institute for Developmental Research, 2. Center for medical genetics, Dept Med, Keio Univ, 3. Aichi Developmental Disability Center, Central Hospital
Keyword: microcephaly, CEP152, GONAD, centrosome
Centrosomal protein 152 (CEP152) is a core centrosomal protein composed of multiple predicted coiled-coil domains. While CEP152 acts as a scaffold to ensure accurate centrosome duplication during cell division, it is also involved in cell polarity and motility. CEP152 gene abnormalities are responsible for autosomal recessive primary microcephaly and Seckel syndrome. Trio-based whole exome sequencing identified novel compound heterozygous variations, c.314G>A/p.(Trp105*) and c.2689A>T/p.(Lys897*), in CEP152 in a Seckel syndrome patient. Cell biological characterization revealed that CEP152-Lys897* was abnormally distributed in the cytosol while CEP152-Trp105* was not detected perhaps due to degradation both in Neuro2A cells in vitro and cortical neurons in vivo. Subcellular localization at the centrosome of the Plk4, a CEP152-binding partner required for centriole formation, was abrogated by co-expression of CEP152-Lys897* in primary hippocampal neurons. We then generated a mouse model mimicking the gene abnormalities of the patient using iGONAD (improved Genome-editing via Oviductal Nucleic Acids Delivery) method. The CEP152-deficient mice recapitulated the clinical features of Seckel syndrome, such as microcephaly, growth impairment, anemia and hypoplasia of testes. Abnormal mitotic spindle pole number was observed in dividing cells in the developing neocortex and cerebellum of the deficient mouse. In addition, CEP152-deficient neuronal precursors and spermatogonia exhibited progressive cell death in vivo. Collectively, the reduction in brain size and short stature observed in the present patient are considered to associate with impaired centrosome duplication, altered subcellular localization of PLK4 and subsequent cell death in proliferative tissues. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-106
有胎盤類特有のzip code配列獲得による放射状グリア細胞内mRNA輸送機構の進化
Evolution of an mRNA transport mechanism in radial glial cells by acquisition of a unique zip code sequence in placental mammals
*吉川 貴子(1)、若松 義雄(1)、井上-上野 由紀子(2)、鈴木 久仁博(3)、井上 高良(2)、大隅 典子(1)
1. 東北大学大学院医学系研究科 発生発達神経科学分野、2. 国立精神・神経医療研究センター 神経研究所 疾病研究第６部、3. 日本大学 松戸歯学部 口腔科学研究所
*Takako Kikkawa(1), Yoshio Wakamatsu(1), Yukiko U. Inoue(2), Kunihiro Suzuki(3), Takayoshi Inoue(2), Noriko Osumi(1)
1. Dept Dev Neurosci, Grad Sch Med, Tohoku Univ, 2. Dept Biochem Cell Biol, NCNP, 3. Res Inst Oral Sci, Nihon Univ, Sch Dent Matsudo
Keyword: cortex, radial glial cells, mRNA transport, cell cycle
Radial glial (RG) cells, the stem/progenitor cells in the developing cortex, are apico-basally polarized, and have long basal processes. We have shown in mouse that mRNAs of Cyclin D2 (Ccnd2), a gene coding a cell cycle regulator, are transported to the basal end-foot of the RG cell, and its 3´UTR is sufficient for this transport as a “zip code” (cis-acting transport element, CTE; Tsunekawa et al., EMBO J, 2012). In this study, we removed the CTE with CRISPR/Cas9 system in mice. In the CTE-deleted mutant cortex, Ccnd2 mRNA no longer localized in the basal end-feet, but accumulated in the RG cell soma, indicating that the CTE is essential for this basal transport. While the Ccnd2 mRNAs are known to be locally translated in the basal end-feet of RG cells, it was unclear whether Ccnd2 proteins in the basal end-feet would be transported to their nuclei, as shown in other proliferating cell types. Using the combination of photo-conversion and time-lapse imaging, we found a basal-to-apical translocation of the photo-converted exogenous Ccnd2 proteins along the basal processes. This result suggests that Ccnd2 proteins in the basal end-feet will be actively transported to the nuclei to modulate the cell cycle progression. Interestingly, the CTE is only conserved in placental mammals among various vertebrates. Consistently, Ccnd2 mRNAs were not detected in the basal end-feet of the opossum, a marsupial mammal, nor in chick. Furthermore, we electroporated an EGFP reporter construct carrying mouse Ccnd2 3’UTR into RG cells of opossum and chick, and found that the mRNA was transported to their end-feet, suggesting that the transport machinery is evolutionarily conserved in mouse, opossum, and chick. As Tbr2-positive proliferative basal progenitors are only present in the placental mammals, and as the number of such progenitors was significantly decreased in the CTE-deleted mutant mice, the basal transport of Ccnd2 mRNA in the RG cells might be involved in producing basal progenitors to make a larger cortex of placental mammals. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-107
Drp1のSUMO化によるミトコンドリア形態制御を介した大脳皮質発生機構
Drp1 SUMOylation regulates cortical development via mitochondrial dynamics
*山田 晴也(1)、佐藤 彩佳(1)、石原 直忠(2)、秋山 博紀(3)、榊原 伸一(1)
1. 早稲田大学人間科学学術院分子神経科学研究室、2. 大阪大学理学研究科細胞生命科学研究室、3. 早稲田大学人間総合研究センター
*Seiya Yamada(1), Ayaka Sato(1), Naotada Ishihara(2), Hiroki Akiyama(3), Shin-ichi Sakakibara(1)
1. Lab Mol Neuro Bio, Grad Sch Hum Sci, Waseda Univ, 2. Dep Biol Sci, Grad Sch Sci, Osaka Univ, 3. Adv Res Cent Hum Sci, Waseda Univ
Keyword: SUMO, Senp, mitochondria, corticogenesis
SUMOylation is a reversible post translational modification. Covalent conjugation of small ubiquitin-like modifier (SUMO) regulates the stability and function of the target protein. SUMOs are then removed from the substrates by sentrin/SUMO-specific proteases (Senps). Numerous studies have implicated that the balance of those enzymes governing SUMOylation and de-SUMOylation are crucial for various physiological and pathological processes in the central nervous system. A mitochondrion is a highly dynamic organelle that undergoes fission and fusion. Dynamin-related protein 1 (Drp1) has a crucial role in the mitochondrial fission machinery. SUMOylation/deSUMOylation controls mitochondrial dynamics through Drp1 modulation. However, the molecular mechanism of Drp1 SUMOylation-mediated mitochondrial dynamics remains unclear. Moreover, we find no reports showing the detailed machinery for SUMOylation-dependent regulation of cerebral cortex development.
Here, we report that the novel Senp5 isoform (Senp5S), which lacks peptidase activity, prevents Drp1 deSUMOylation in a competitive manner (Yamada et al., iScience, 2021). In addition, we found Senp5S and conventional full-length Senp5 (Senp5L) regulate mitochondrial morphology through regulating SUMOylation/deSUMOylation of Drp1. Moreover, Senp5 perturbations in E14.5 embryonic cortex using in utero electroporation repressed the proper migration of neurons, leading the accumulation of newborn neurons in the intermediate zone. Accordingly, we investigated the physiological roles of Senp5 on the cerebral cortex development. Forced expression or knockdown of Senp5L/5S in the primary cultured neurons resulted in the reduced number of neurites and perturbation of the axonal extension.
These findings suggest a novel role of post-translational modification, in which deSUMOylation enzyme isoforms competitively regulate mitochondrial dynamics via Drp1 SUMOylation levels, in a tightly controlled process of neuronal differentiation and corticogenesis. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-108
生後発達におけるサブプレートニューロンのダイナミクス
Dynamics of Subplate neurons in postnatal development
*守屋 敬子(1)、杉田 祐輔(1,2)、高橋 淑子(1)、丸山 千秋(1)
1. （公財）東京都医学総合研究所、2. 東京理科大学先進工学部
*Keiko Moriya-Ito(1), Yusuke Sugita(1,2), Yoshiko Takahashi(1), Chiaki Ohtaka-Maruyama(1)
1. Tokyo Met Ins of Med Sci, Tokyo, Japan, 2. Tokyo Univ of Sci, Tokyo, Japan
Keyword: subplate , cerebral cortex, birthdate analysis
The Subplate layer is a cytoarchitectural compartment between the cortical plate and intermediate zone in the developing neocortex and mainly consist of subplate neurons (SpNs) and extracellular matrix (ECM). SpNs play critical roles in forming correct cortical layers and thalamocortical circuits in the developing brain. They are one of the earliest born neurons, and most of SpNs disappear until the early postnatal period. In contrast, recent reports suggest that SpNs survive throughout life, especially in primates and play roles in normal cortical functions. Moreover, it also reports that SpNs are a heterogeneous cell population in terms of their gene expression and functions. However, which subtypes are more likely to survive for more extended periods after birth remains elusive. Our goal is to understand the functions of SpNs throughout life. We first confirmed whether the earliest born SpNs exist in postnatal developing mouse brains. SpNs born at embryonic day10 and 11 were visualized using BrdU or EdU injection and counted in the subplate layer, located between the white matter and cortical layer 6a. The BrdU- or EdU-labeled SpNs remarkably decreased until postnatal day10 and gradually diminished as age advanced. Moreover, we found that 20% or more of NeuN-positive SpNs were BrdU positive in the subplate layer in the adult mouse brains. This result supports the theory of persistence of the adult SpNs in mice. Next, to clarify the lineage property of surviving SpNs, we used Lpar1-GFP transgenic mice in which a part of SpNs genetically labelled with GFP. Lpar1-GFP positive SpNs were localized along the external capsule (EC)at postnatal day 0, and approximately 30 % of neurons in the subplate layer were GFP positive. A certain number of SpNs remained in the adult. However, the survival rates of GFP expressing SpNs were different between cortical areas. It was higher in the motor and the sensory cortex than in the visual and the auditory cortex. Furthermore, we investigate the relationship between SpNs linage, birthdate, and survival. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-109
哺乳類大脳皮質発生で、サブプレートニューロンが皮質板下に配置されるメカニズム
How are the subplate neurons positioned below the cortical plate in the developing mammalian cortex?
*吉永 怜史(1,2)、石井 一裕(2)、岡本 麻友美(3)、宮田 卓樹(3)、久保 健一郎(1,2)、仲嶋 一範(2)
1. 東京慈恵会医科大学、2. 慶應義塾大学医学部、3. 名古屋大学大学院医学系研究科
*Satoshi Yoshinaga(1,2), Kazuhiro Ishii(2), Mayumi Okamoto(3), Takaki Miyata(3), Ken-ichiro Kubo(1,2), Kazunori Nakajima(2)
1. Jikei Univ Sch Med, Tokyo, Japan, 2. Keio Univ Sch Med, Tokyo, Japan, 3. Grad Sch Med, Nagoya Univ, Nagoya, Japan
Keyword: SUBPLATE, CORTICAL DEVELOPMENT, CYTOARCHITECTURE, WHITE MATTER NEURONS
In the developing mammalian cerebral cortex, the earliest-generated subplate (SP) neurons first reside in the preplate (PP), which is split into a superficial layer called the marginal zone (MZ), and a deep layer, the SP, upon the formation of the cortical plate (CP). SP neurons contribute to white matter neurons, which are increased in some patients with psychiatric disorders. The subplate specific genes during development also show an enrichment for association with these disorders. Some reports suggested that SP neurons are positioned below the CP passively since “layer VI neurons” split the PP. However, it has not been experimentally verified whether the neurons that split the PP are the layer VI neurons, and how the SP layer is properly formed remains to be elucidated. Here, we specifically labeled future SP neurons with FlashTag technology, which labels cells that undergo mitosis on the ventricular surface with high temporal resolution, to describe migratory and positional profiles. Labeled cells were first observed in the PP and then in the CP and MZ upon the formation of the CP; finally, they moved down below the CP 1-2 days later, suggesting downward movement of some SP neurons through the CP. This observation was independently validated by ontogenetic observations using in utero electroporation, BrdU incorporation, immunohistochemistry, and in situ hybridization. Furthermore, SP neuron-specific expression of a dominant negative form of PI3-kinase resulted in abnormal positioning above and below the SP, which was rescued by expression of a constitutive active form of Rheb. These observations suggest that future SP neurons actively migrate to form a distinct layer below the CP in a PI3-kinase signaling-dependent manner. These observations may provide new insights for understanding cellular mechanisms of vulnerability to, and pathogenesis of, psychiatric disorders. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-110
シングルセルトランスクリプトームに基づくホヤ幼生神経索グリア細胞の神経発生および神経機能における役割の解析
Analysis of developmental and physiological roles of the nerve cord glial ependymal cells in the ascidian larva based on single-cell transcriptomes
*圓尾 綾菜(1)、曽谷 実玖(2)、大川 奈菜子(2)、堀江 健生(3)、日下部 岳広(1,2)
1. 甲南大学理工学部・統合ニューロ、2. 甲南大学大学院自然科学研究科、3. 筑波大学下田臨海実験センター
*Ayana Maruo(1), Miku Sotani(2), Nanako Okawa(2), Takeo Horie(3), Takehiro G. Kusakabe(1,2)
1. Dept Biol & Inst Integr Neurobiol, Konan University, 2. Grad Sch Nat Sci, Konan University, 3. Shimoda Mar Res Cntr, University of Tsukuba
Keyword: ascidian, glia, ependymal cell, floor plate
The central nervous system of the ascidian larva is divided into three parts, a brain vesicle, a motor ganglion, and a caudal nerve cord, from anterior to posterior. The brain vesicle contains sensory organs and diverse interneurons, which presumably form neural circuits for sensory information processing and motor regulation. The motor ganglion/nerve cord, in which Hox genes are expressed, has been suggested to be homologous to the vertebrate hindbrain/spinal cord. The motor ganglion contains cholinergic motor neurons that control contraction of the tail muscle cells for swimming. The hollow nerve cord mainly consists of four rows of glial ependymal cells: rows of the dorsal roof plate cells, the left and right lateral wall cells, and the ventral floor plate cells. Compared to the sensory organs and neurons of the brain vesicle and motor ganglion, the developmental and physiological roles of the ependymal cells of the nerve cord remain elusive. To understand developmental and physiological roles of the nerve cord ependymal cells, we performed single-cell transcriptomic profiling of each row of the nerve cord cells in the ascidian Ciona intestinalis type A (Ciona robusta). The three types of the ependymal cells (roof plate, lateral walls, and floor plate) are clearly distinguished by the transcriptome profiles. For example, the floor plate cells specifically express hedgehog.b and fgf3/7/10/20, the roof plate cells express wnt7 and msx.b, and the lateral wall cells express gnrh2 and tbx2. Identified ependymal cell-specific genes encode proteins involved in regulation of nervous system development and physiological functions, including molecules related to ciliary functions, axon guidance, cell-cell interaction, and intracellular signaling. In addition, the single-cell transcriptomic analysis revealed conspicuous diversity of ependymal cells along the anteroposterior axis. For example, two distinct isoforms of ephrin ligands, known to act as repulsive cues in axon guidance, are expressed in specific regions of the lateral walls and the floor plate along the anteroposterior axis. We discuss developmental and physiological roles of the glial ependymal cells of the Ciona nerve cord based on the gene expression profiles and experimental manipulation of selected genes. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-111
ヒストンメチル化酵素Setd8は網膜発生において網膜前駆細胞の増殖を制御する
Histone lysine methyltransferase Setd8 regulates proliferation of retinal progenitor cells during retinal development
*石龍 悠(1)、松原 周蔵(1)、小田 尚伸(2)、松田 泰斗(1)、中島 欽一(1)
1. 九州大学大学院医学研究院基盤幹細胞学分野、2. 済生会熊本病院 総合腫瘍科がんゲノムセンター
*Haruka Sekiryu(1), Shuzo Matsubara(1), Hisanobu Oda(2), Taito Matsuda(1), Kinichi Nakashima(1)
1. Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 2. Saiseikai Kumamoto Hospital, Division of integrative medical oncology, Department of cancer genome medicine
Keyword: Setd8, H4K20me1, retina
Five major types of neurons (photoreceptors, bipolar cells, horizontal cells, amacrine cells, ganglion cells) and one type of glial cells (Müller glia) are generated from common retinal progenitor cells (RPCs) in a chronologically defined order during retinal development. It is generally known that epigenetic modifications, including histone modifications, play critical roles in fate specification of a variety of stem/progenitor cells. Setd8 is the sole mammalian enzyme, which catalyzes monomethylation of histone H4 at lysine 20 (H4K20me1) and is essential for mammalian development since targeted disruption of Setd8 and subsequent loss of H4K20me1 result in pre-implantation lethality. However, the role of Setd8 during retinal development remains elusive. We, therefore, sought to examine whether Setd8 regulates the behavior of RPCs during mouse retinal development. We first confirmed that H4K20me1 modification exists in RPCs from the embryonic stage to early postnatal stages, implying that Setd8 plays some role in RPCs. Conditional knockout of Setd8 in RPCs at embryonic day (E) 17 reduced the number of bipolar cells and Müller glia when we examine the retina at postnatal day (P) 15, although differentiation of RPC into these two cell types was not affected. This result led us to hypothesize that the deletion of Setd8 decreases RPC proliferation, resulting in the decreased number of these differentiated cells. To test this idea, we performed Setd8 knockdown in RPCs in vitro and observed the inhibition of RPC proliferation without inducing cell death. Furthermore, when we cultured RPCs in the presence of a selective Setd8 inhibitor UNC0379, which reduces the levels of H4K20me1 by inhibiting Setd8, proliferation of the RPCs was inhibited. Taken together, our findings suggest that Setd8 positively regulates RPC proliferation without affecting retinal cell differentiation and survival of RPCs, and contributes to proper retinal development. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-112
Dab1のハプロ不全は大脳新皮質Layer1厚減少と尾側海馬CA1錐体細胞層の分離を引き起こす
Haploinsufficiency of Dab1 causes reduction of neocortical layer 1 thickness and splitting of caudal hippocampal CA1 pyramidal cell layer
*本田 岳夫(1,2)、廣田 ゆき(1)、仲嶋 一範(1)
1. 慶應義塾大学医学部解剖学、2. 岐阜薬科大学薬学部分子生物学
*Takao Honda(1,2), Yuki Hirota(1), Kazunori Nakajima(1)
1. Dept Anat, Sch Med, Keio Univ, Tokyo, Japan, 2. Lab Mol Bio, Gifu Pharm Univ, Gifu, Japan
Keyword: Reelin-Dab1 signaling, layer formation, apical dendrite, neuronal migration
Cerebral cortex plays a critical role in higher brain functions such as perception and cognition. For the development of the cerebral cortex, REELIN and DAB1 signaling pathway is essentially important. Lack of these genes cause severe defects in neuronal positioning in the cerebral cortex. However, the mechanisms mediating this function remains still elusive. Here we show that Dab1 haploinsufficiency causes half reduction of layer1 thickness and slight migration delay of hippocampal neurons. Using wild-type and heterozygous yotari mice, an autosomal recessive mutant of Dab1, we found that, although there were no obvious neuroanatomical differences between the genotypes at postnatal day (P) 0, heterozygous yotari mice at P7 had a thinner layer 1 in the cerebral neocortex compared with that of wild-type mice. The reduction of layer 1 thickness was associated with superficial shift of the positioning of layer 2-4 neurons. Birthdating study suggested that reduction of layer 1 in the cerebral neocortex was not caused by failure of migration. In utero electroporation-mediated sparse labeling of the most superficial layer neurons of cerebral neocortex showed that the superficial layer neurons of heterozygous yotari mice tend to elongate their apical dendrites within layer 2 than within layer 1. As to hippocampus, we found that the caudo-dorsal part of the CA1 pyramidal cell layer is abnormally split in heterozygous yotari mice. Birthdating study revealed that splitting of CA1 pyramidal cell layer is mainly caused by migration failure of the late-born pyramidal neurons in the caudal part of hippocampus. Recombinant adeno-associated virus-mediated sparse labeling of hippocampal pyramidal cells showed that pyramidal cells within the split cell layer tend to have misguided apical dendrites. These results raise a possibility that cerebral neocortex have two distinct DAB1-related pathways in terms of their dependency on the dosage of Dab1 gene, which is haplosufficient for the regulation of neuronal migration but haploinsufficient for the control of layer 1 thickness. As to hippocampus, migration of a subpopulation of pyramidal neurons is thought to be sensitive to the half reduction of the Dab1 gene. These results will help dissect the REELIN-DAB1 signaling pathways to understand how this signaling controls multiple aspects of cortical development in a context-dependent manner. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-113
多能性幹細胞由来原始マクロファージならびにドパミン神経細胞の誘導におけるニコチン性アセチルコリン受容体の機能解析
Roles of nicotinic acetylcholine receptors on the differentiation of pluripotent stem cell-derived primitive macrophages and dopaminergic neurons
*原田 考輝(1)、加藤 丈使(1)、平尾 真大(1)、西村 周泰(1)、下濱 俊(2,3,4)、高田 和幸(1)
1. 京都薬科大学統合薬科学系、2. 札幌医科大学神経内科学講座、3. 慈誠会認知症センター、4. 慈誠会記念病院
*Koki Harada(1), Takeshi Kato(1), Masahiro Hirao(1), Kaneyasu Nishimura(1), Shun Shimohama(2,3,4), Kazuyuki Takata(1)
1. Div. Integ. Pharm. Sci., Kyoto Pharm. Univ., Kyoto, Japan, 2. Dept. Neurol., Sapporo Med. Univ. Sch. of Med., Sapporo, Japan, 3. Jiseikai Dementia Ctr., Tokyo, Japan , 4. Jiseikai Mem. Hosp., Tokyo, Japan
Keyword: nicotinic acetylcholine receptors, microglia, dopaminergic neurons, pluripotent stem cell
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are widely expressed in a variety of cells from embryonic stage to adult. nAChRs are known to be involved in the pathophysiology of brain diseases, such as Alzheimer's disease and Parkinson's disease. Elucidation of the roles of nAChRs will help to clarify the pathogenesis and therapeutic strategies. However, the roles of nAChRs on the differentiation and development of brain cells, such as microglia and dopaminergic (DA) neurons, have not been fully elucidated. In this study, we analyzed the expression patterns of nAChR subunits during the development of primitive macrophages, which are microglia precursor cells, and DA neurons and clarified the roles of nAChRs on the differentiation processes from pluripotent stem cells. Mouse embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells were used for the derivation of mouse primitive macrophages and human DA neurons, respectively. The expression of nAChR subtypes was analyzed by RT-PCR at multiple time points during differentiation process. Effects of nAChR agonists on the expression of mRNAs and proteins were clarified by quantitative PCR and flow cytometry, respectively. Various nAChR subunits, such as α2, α4, α6 and α7 subunits, were expressed during differentiation processes. In the early stage of the differentiation of primitive macrophage, nicotine, a non-specific nAChRs agonist, increased the differentiation efficiency from ES cells to primitive macrophage progenitor cells (Flk-1+ cells). Furthermore, expression levels of CX3CR1 protein, a chemokine receptor, was increased by nicotine treatment at the terminal differentiation stage. On the other hand, treatment with nicotine during early stage of neuronal differentiation tended to increase the expression of FOXA2 and LMX1A, markers of DA neuron progenitors. Stimulation with selective agonist of α6 nAChRs at the neural maturation stage accelerated differentiation to DA2 neurons expressing LMO3, which are a subpopulation of DA neurons projecting from the substantia nigra pars compacta to the striatum. These results suggest that nAChRs play important roles in the differentiation, maturation, and function of primitive macrophages and DA neurons. This study may provide deep insights into the developmental mechanisms of microglia and DA neurons and the role of nAChRs and contribute to the development of nAChR-targeted therapeutic strategies for brain diseases. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-114
生後ラットの血液脳関門形成とグリア細胞の関与
Developmental formation of blood brain barrier in rat brains and the role of glial cells.
*最上（重本） 由香里(1,2)、北村（中山） 貴美子(1)、佐藤 薫(1)
1. 国立医薬品食品衛生研究所 薬理部 、2. 山梨大学 医学部 薬理学講座
*Yukari Shigemoto Mogami(1,2), Kimiko Nakayama Kitamura(1), Kaoru Sato(1)
1. Dept. Neuropharmacol. NIHS, 2. Dept. Neuropharmacol. Grad. Sch. Medicine, Yamanashi Univ.
Keyword: Blood brain barrier, microglia, Astrocyte, developmental
Blood vessels in the central nervous system (CNS) have a strong barrier function called the blood brain barrier (BBB), which enables the CNS-specific pharmacokinetics. Recent studies have clarified that the vascular endothelial cells and the surrounding cells control the formation and maturation of BBB. However, the period of BBB formation and the underlying mechanisms remain to be elucidated. In this study, we investigated the period of cerebrovascular BBB formation in the rat brain using Evans Blue or a biotin-labeled reagent, that do not pass through maturated BBB. We found that rat cerebrovascular BBB was formed between day 4 and day 10 after the birth. The expression of proteins important for BBB-specific functions were increased during the BBB formation process. Moreover, we observed that during BBB formation the capillaries were surrounded by astrocyte endfeets and microglia. Of interest, microglia directly contacted with the capillaries. Currently we are investigating the concrete contributions of microglia in BBB formation and maturation. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-115
大脳皮質神経幹細胞の発生段階依存的性質変化を制御するエピジェネティクス機構の解明
Epigenetic mechanisms regulating developmental stage-dependent change in cortical neural stem cell property.
*中川 拓海(1)、今村 拓也(2)、堅田 明子(1)、中島 欽一(1)
1. 九州大学院医学系学府、2. 広島大学院統合生命科学研究科
*Takumi Nakagawa(1), Takuya Imamura(2), Sayako katada(1), Kinichi Nakashima(1)
1. Grad Sch Med Sci, Univ of Kyushu, Fukuoka, Japan, 2. Grad Sch Integrated Sci for life, Univ of Hiroshima, Hiroshima, japan
Keyword: Cortical development, Trim28, DNA methylation, histone modification
During the development of mammalian cerebral cortex, neural stem cells (NSCs) produce only neurons in mid gestation, and subsequently acquire the ability to give rise to glial cells (astrocyte and oligodendrocyte). This temporal switch of NSCs property along developmental stage is regulated by epigenetic modifications such as DNA methylation and histone modifications. For example, DNA demethylation in astrocyte-related genes and methylation of histone H3 lysine (K)27 in neuron-related genes are important for the acquisition of astrocytic differentiation potential. In this relation, we have recently identified gained DNA methylation regions (GDMRs) close to neuron-related genes in NSCs with the progression of development, however, its effects and relevant factors on differentiation potential of NSCs remain elusive. In this study, we identified factors that may bind to these GDMR, and analyzed their functions implicated in NSC property change. First, we searched for transcription factors that bind to GDMRs by using publicly available ChIP-seq data. Since we found that the expression of genes adjacent to GDMRs is repressed, we choose a transcriptional repressor Trim28, which is highly expressed in NSCs compared to other identified candidate factors. Next, we knocked down Trim28 in NSCs prepared from relatively late embryonic stage (E14), and observed that the expression of a proneural gene Neurog1 was increased, promoting neuronal differentiation of the NSCs. Analysis of some epigenetic modifications around the Neurog1 promoter revealed an increase and decrease in H3K27 acetylation and methylation, respectively, while DNA methylation level was not altered by Trim28 knockdown. Although Neurog1 was identified as a gene which is close to GDMR, these results suggest that Trim28 H3K27 modification dependently but DNA methylation-independently suppresses Neurog1 expression and eventually neuronal differentiation. Given that SUMOylated Trim28 is known to interact with the chromatin remodeling factor NuRD complex to deacetylate H3K27, we then examined whether Trim28 is highly SUMOylated in late embryonic (E14) NSCs, and found it is the case. Therefore, we are currently exploring the function of Trim28 in NSC fate regulation from the viewpoint of histone acetylation-related mechanism. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-116
哺乳類大脳皮質の発生と進化におけるGsk3βの細胞種特異的な転写制御
Cell type-specific transcriptional control of Gsk3β in the development and evolution of the mammalian neocortex
*野村 真(1)、後藤 仁志(1)、清成 寛(2)、 小野 勝彦(1)
1. 京都府立医科大学、2. 理化学研究所生命機能開発センター
*Tadashi Nomura(1), Hitoshi Gotoh(1), Hiroshi Kiyonari(2), Katsuhiko Ono(1)
1. Kyoto Prefectural University of Medicine, 2. RIKEN BDR
Keyword: Neocortex, Wnt signaling, Gsk3β, Neurogenesis
Temporal control of neurogenesis is central for the development and evolution of species-specific brain architectures. The balance between progenitor expansion and neuronal differentiation is tightly coordinated by cell-intrinsic and cell-extrinsic cues. Wnt signaling plays pivotal roles in the proliferation and differentiation of neural progenitors in a temporal manner. However, regulatory mechanisms that adjust intracellular signaling amplitudes according to cell fate progression remain to be elucidated. Here, we report the transcriptional controls of Gsk3β, a critical regulator of Wnt signaling, in the developing mouse neocortex. Gsk3β expression was higher in ventricular neural progenitors, while it gradually declined in differentiated neurons. We identified active cis-regulatory module (CRM) of Gsk3β that responded to cell type-specific transcription factors, such as Sox2, Sox9 and Neurogenin2. Furthermore, we found extensive conservation of the CRM among mammals but not in nonmammalian amniotes, in line with the species differences in Gsk3β expression in reptilian and avian brains. Our data suggest that a mammalian-specific CRM drives the cell type-specific activity of Gsk3βto fine tune Wnt signaling, which contributes to the tight control of neurogenesis during neocortical development and evolution. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-117
アルギニンメチル化酵素CARM1によるオリゴデンドロサイト分化制御
Coactivator-associated arginine methyltransferase 1 controls oligodendrocyte differentiation in the corpus callosum during brain development
*石野 雄吾(1)、清水 尚子(1)、遠山 正彌(1)、宮田 信吾(1)
1. 近畿大学東洋医学研究所
*Yugo Ishino(1), Shoko Shimizu(1), Masaya Tohyama(1), Shingo Miyata(1)
1. Kindai University
Keyword: Arginine methylation, PRMT, oligodendrocytes, myelination
Protein arginine methylation has been recognized as one of key post-translational modifications for refined protein functions, mediated by protein arginine methyltransferases (Prmts). Coactivator-associated arginine methyltransferase (Carm1, also known as Prmt4) participates in various cellular events, such as cell survival, proliferation, and differentiation through its protein arginine methylation activities. Carm1 regulates cell proliferation of a neuronal cell line and is reportedly expressed in the mammalian brain. However, its detailed function in the central nervous system, particularly in glial cells, remains largely unexplored. In this study, Carm1 exhibited relatively high expression in oligodendrocyte (OL) lineage cells present in the corpus callosum of the developing brain, followed by a remarkable downregulation after active myelination. The suppression of Carm1 activity by inhibitors in isolated oligodendrocyte precursor cells (OPCs) reduced the number of Ki67-expressing and BrdU-incorporated proliferating cells. Furthermore, Carm1 inactivation attenuated OL differentiation, as determined by the expression of Plp, a reliable myelin-related marker. It also impaired the extension of OL processes, accompanied by a significant reduction in gene expression related to OL differentiation and myelination, such as Sox10, Cnp, Myrf, and Mbp. In addition, OLs co-cultured with embryonic dorsal root ganglia neurons demonstrated that Carm1 activity is required for the appropriate formation of myelin processes and myelin sheaths around neuronal axons, and the induction of the clustering of Caspr, a node of Ranvier structural molecule. Thus, we propose that Carm1 is an essential molecule for the development of OPCs and OLs during brain development. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-118
マウス海馬CA1と大脳新皮質錐体細胞は発生期において異なる移動様式を示す
Mouse hippocampal and neocortical neurons demonstrate different migration modes during brain development
*北澤 彩子(1,2)、吉永 怜史(1,2)、シン ミンギョン(2,3)、林 周宏(2)、佐野 ひとみ(2,4)、大石 康二(2,5)、久保 健一郎(1,2)、仲嶋 一範(2)
1. 東京慈恵会医科大学解剖学講座、2. 慶應義塾大学医学部解剖学教室、3. KBRI、4. システム・バイオロジー研究機構、5. 同志社大学大学院脳科学研究科
*Ayako Kitazawa(1,2), Satoshi Yoshinaga(1,2), Minkyung Shin(2,3), Kanehiro Hayashi(2), Hitomi Sano(2,4), Koji Oishi(2,5), Ken-ichiro Kubo(1,2), Kazunori Nakajima(2)
1. Dept of Anat, Univ of Jikei, Tokyo, Japan, 2. Dept of Anat, Univ of Keio, Tokyo, Japan , 3. KBRI, Daegu, Korea, 4. SBI, Tokyo, Japan, 5. Grad Sch of Brain Sci, Univ of Doshisha, Kyoto, Japan
Keyword: hippocampus, neocortex, neuronal migration, transplantations
Most excitatory neurons in the neocortex and hippocampus are born near the ventricle and migrate to their final destinations. Neuronal migration is essential for the formation of brain structures during development, and disruption of normal migration would be the cause of brain malformation in several human diseases and mouse mutants. To date, the analyses of the neuronal migration have mainly been performed for the neocortex. On the other hand, despite the importance of hippocampal functions, cellular and molecular mechanisms of its development are not yet fully understood. We previously reported that the hippocampal CA1 pyramidal neurons, unlike the neocortical neurons, migrate in a zigzag manner with highly branched processes using multiple hippocampal radial fibers as scaffolds (climbing mode of migration). In this study, we searched for the factors that determine neuronal migration modes, i.e., locomotion mode in the neocortex or climbing mode in the hippocampus, by using cell transplantation and a newly developed cell-reduced culture system. The results suggested that the differences in the migration mode between the neocortex and hippocampus were not primarily caused by different external environment around the migrating cells but could mainly be attributable to the distinct properties of migrating cells themselves. Furthermore, we carried out microarray analysis to find genes expressed differentially between neocortical and hippocampal migrating neurons, and are now investigating their potential involvement in determining the distinct migration modes in the neocortex and hippocampus. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-119
磁場照射マウスとABCA1欠損マウスでの歯状回における成体脳海馬神経新生の制御
Adult-neurogenesis at the hippocampal dentate gyrus is reduced in the magnetic field treated wild-type mice, and in untreated Abca1-null mice
*辻田 麻紀(1)、高瀬 弘嗣(1)、古家 圭人(2)、鍔木 基成(2,3)、熊本 奈都子(1)、鵜川 眞也(1)
1. 名古屋市立大学大学院医学研究科 、2. 神戸大学大学院理学研究科、3. 神戸大学研究基盤センター
*Maki Tsujita(1), Hiroshi Takase(1), Keito Furuie(2), Motonari Tsubaki(2,3), Natsuko Kumamoto(1), Shinya Ugawa(1)
1. Nagoya City University Graduate School of Medical Sciences, 2. Graduate School of Science and Technology, Kobe University, 3. Research Facility Center for Science and Technology, Kobe University
Keyword: magnetic field, dentate gyrus, ABCA1, Click Chemistry
Aim: This project aims to explore the role of magnetic field (MF) exposure on cholesterol metabolism. Method: Male 8-week-old C57BL/6N mice were administered F-ara-EdU/PBS (133 micro-g/g mouse) intraperitoneally and exposed to a magnetic field (MF), 0.4 T for 2 h, using an EPR spectrometer. Control wild-type mice and Abca1-null mice were kept under the same conditions in the absence of MF exposure. Mouse brains were collected on day 28 post-exposure and 40 μm coronal sections prepared with a Leica CM1900 cryostat. F-ara-EdU in the sections were labeled with Alexa-488 using the free-floating Click Chemistry method. Hippocampal dentate gyrus (DG) images were observed with a Spin SR10 and the OlyVIA application (Olympus). Results: Newborn cells incorporated with F-ara-EdU at the DG were observed. In wild-type control mice, an average of 1.6 cells per DG slice were found in the granular zone and in the subgranular zone. In contrast, MF-exposed mice had 0.47 positive cells per DG slice (P = 0.003). Similar to the MF-exposed wild-type mice, 0.44 positive cells per DG slice (P=0.0001 vs control) were detected in Abca1-null mouse DG. The ABCA1-apoA-I-mediated HDL generation from mouse peritoneal macrophage foam cells was reduced by 0.4 T MF exposure. In addition, the lipoprotein profile in the blood of mice exposed to MF showed a decrease in HDL cholesterol levels even after 28 days. Conclusions: Newly generated brain cells at the hippocampal DG, detected using F-ara-EdU, were significantly reduced by MF exposure to the same extent as the newly generated brain cells in the DG of Abca1-null mice. ABCA1 is one of the factors where expression is increased by retinoic acid which regulates the early stage of adult neurogenesis. In this study, ABCA1-deficient mice showed the same decrease as that of mice exposed to MF. It is likely that ABCA1-dependent HDL genesis, impaired by MF exposure during the early stages of adult neurogenesis, is the one of causes. Since apoA-I is the most efficient ligand for ABCA1, we will examine the adult neurogenesis in mice lacking apoA-I in future studies. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-120
大脳新皮質第4層発達過程の時空間的遺伝子発現と統合ダイナミクス
Spatiotemporal gene expression and integration dynamics of neocortical layer 4 neurons revealed by temporal genetic fate-mapping
*大手 直人(1,2)、侯 珮珊(2,4,5)、西山 千尋(4)、花嶋 かりな(1,2,3,4)
1. 早稲田大学教育学部理学科生物学専修、2. 早稲田大学大学院先進理工学研究科生命理工学専攻、3. 早稲田大学教育・総合科学学術院、4. 理化学研究所大脳皮質発生研究チーム、5. 陽明交通大學解剖學及細胞生物學研究所
*Naoto Ohte(1,2), Pei-Shan Hou(2,4,5), Chihiro Nishiyama(4), Carina Hanashima(1,2,3,4)
1. Faculty of Education, Department of Science, Waseda University, Tokyo, Japan, 2. Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan, 3. Faculty of Education and Integrated Arts and Sciences, Waseda University, Tokyo, Japan, 4. Laboratory for Neocortical Development, RIKEN Center for Developmental Biology, Kobe, Japan, 5. National Yang Ming Chiao Tung University Institute of Anatomy and Cell Biology, Taipei, Taiwan
Keyword: Cerebral cortex, Layer 4
The cerebral cortex, which covers the surface of the cerebrum, consists of 6 neuronal layers that assemble complex cortical circuits that are physiologically and mentally indispensable for higher order information processing. During the prenatal period, cortical neurons are produced from neural stem cells within the ventricular zone that sequentially produce deep- and upper-layer neurons. These neurons migrate radially towards the surface and later born neurons overtake early born neurons and accumulate in an inside-out manner. Neurons of each layer display morphologically and molecularly unique features, such that layers 2/3 (external granular /pyramidal layer) neurons express Brn2, layer 4 (internal granular Layer) neurons express Rorb, layer 5 (internal pyramidal Layer) neurons express Ctip2 and layer 6 (multiform layer) neurons express Tbr1. Studies over the past decades have revealed core transcriptional regulatory network that confer layers 2/3, 5 and 6 identities, underscoring the importance of intrinsic program in specifying long-range projection neurons. In contrast, it has been considered that the specification and maturation of layer 4 sensory input local projection neurons require extrinsic cues driven via thalamocortical axons in addition to intrinsic mechanisms; however, the relative contribution of these cues in the differentiation of layer 4 neurons remain poorly understood. To explore the molecular logic underlying the differentiation of layer 4 neurons, we utilized genetic fate-mapping by crossing Neurogenin2CreER/+ mice with Rosa26CAG-Loxp-Stop-Loxp-tdTomato reporter mice and administered tamoxifen at specific gestational stages to trace temporal cohorts of glutamatergic projection neurons. These results revealed spatiotemporal gene expression and integration dynamics of layer 4 neuron precursors in the developing cerebral cortex. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-121
HPRTによるミクログリア形態の多様性を生み出す新たなメカニズム
A novel mechanism for morphological diversity of microglia mediated by HPRT
*照屋 林一郎(1)、岡島-高橋 智美(1)、上田 健太郎(2)、鶴田 文憲(1)
1. 筑波大学 生命環境系、2. 筑波大学生命環境学群生物学類
*Rinichiro Teruya(1), Tomomi Okajima-Takahashi(1), Kentaro Ueda(2), Fuminori Tsuruta(1)
1. Grad Sch Life and Env Sci, Univ of Tsukuba, Ibaraki, Japan, 2. Colleg of Biol Sci, Sch of Lif and Env Sci, Univ of Tsukuba, Ibaraki, Japan
Keyword: Microglia, Lesch-Nyhan syndrome, brain development , neuronal circuit system
Microglia, which are immune cells in the central nervous system, play important roles in brain development. Recent research has revealed that microglia have sex differences in their morphology and functions. Previously, we found that an increase of intracellular hypoxanthine concentration controls the microglial morphology in vitro. Hypoxanthine is one of the components of purine bodies and is synthesized as an intermediate of nucleotide metabolisms. It has been reported that the hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) contributes to ATP/GTP synthesis through converting hypoxanthine into inosine monophosphate in the salvage pathway. Mutation in Hprt1 gene causes Lesch-Nyhan syndrome. Lack of this enzyme increases the amount of hypoxanthine. The characteristics defining the disease are not only hyperuricemia as an abnormality of nucleotide metabolisms, but also neurodevelopmental abnormalities and self-injurious behavior. Therefore, it is thought that HPRT activity is important in the developing brain. In this study, we report that HPRT activity related to a morphological change in microglial cell line BV2. In addition, Hprt1 expression has sex differences in microglia in the developmental stage. We found that HPRT expression is upregulated in response to hypoxanthine in BV2 cells. Moreover, we also revealed that HPRT overexpression leads to a morphological change in BV2 cells. We isolated microglia from each sex and examined the expression levels of Hprt1. We found that expression level of Hprt1 in female microglia is higher than that in male microglia. Since Hprt1 gene resides in the X chromosome, it is possible that HPRT is a key gene that controls microglial sex difference and produces microglial diversity. Our data provide the possibility of linking a new function of HPRT in microglia to a critical cause of Lesch-Nyhan syndrome. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-122
妊娠期の偏った多価不飽和脂肪酸摂取は胎仔ドパミンニューロン産生に影響し、仔の様々な行動異常に関連する
Dietary imbalance in polyunsaturated fatty acids during pregnancy affects dopaminergic neurogenesis in their embryos and is associated with offspring's various behavioral abnormalities
*酒寄 信幸(1)、藤井 一希(2)、片倉 賢紀(3)、小林 和人(4)、高雄 啓三(2)、杉田 誠(1)
1. 広島大学大学院医系科学研究科、2. 富山大学学術研究部医学系、3. 城西大学薬学部、4. 福島県立医科大学医学部附属生体情報伝達研究所
*Nobuyuki Sakayori(1), Kazuki Fujii(2), Masanori Katakura(3), Kazuto Kobayashi(4), Keizo Takao(2), Makoto Sugita(1)
1. Grad Sch Biomed Health Sci, Hiroshima University, Hiroshima, Japan, 2. Fac Med, Univ Toyama, Toyama, Japan, 3. Fac Pharmacy Pharmaceutical Sci, Josai Univ, Saitama, Japan, 4. Inst Biomed Sci, Fukushima Med Univ, Fukushima, Japan
Keyword: DOPAMINE, NEUROGENESIS, FATTY ACID, BEHAVIORAL TEST BATTERY
Most animals cannot synthesize omega-6 (n-6) or omega-3 (n-3) polyunsaturated fatty acids (PUFAs), which are essential nutrients for normal brain development and function. In the brain, these PUFAs work as structural components of the cellular membrane and also serve as precursors for bioactive lipid metabolites. Importantly, these PUFAs are generally competitive in various metabolic processes, and thus the n-6/n-3 ratio in our diet warrants particular attention. Based on recent nutritional trends leading to foods that are rich in n-6 PUFAs and poor in n-3 PUFAs, we have previously reported that intake of an n-6-rich/n-3-poor diet by pregnant mice increases midbrain dopaminergic neurogenesis in their embryos and also induces offspring's hedonic feeding behaviors in adulthood. In this study, we investigated how consuming the n-6-rich/n-3-poor diet affects midbrain dopaminergic neurogenesis and also tried to understand comprehensive behavioral phenotypes of the offspring exposed to the n-6-rich/n-3-poor diet in utero. We first confirmed that the brain n-6 and n-3 PUFAs were increased and decreased, respectively, in the embryos exposed to the n-6-rich/n-3-poor diet in utero compared to those exposed to the control diet. We further found that exposure to the n-6-rich/n-3-poor diet increased dopaminergic neurogenesis in the developing midbrain at embryonic day 11.5. Next, we utilized a comprehensive behavioral test battery and revealed that both male and female offspring exposed to the n-6-rich/n-3-poor diet in utero showed a decreased social behavior compared to those exposed to the control diet. We also found that female offspring exposed to the n-6-rich/n-3-poor diet showed hyperactivity compared to those exposed to the control diet. Our findings show that maternal intake of PUFAs during pregnancy can have long-lasting effects on offspring's behaviors in adulthood probably through abnormalities in the process of brain development. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-123
Retrosplenial cortexの特異性に関わる遺伝子と層構造構築過程の解析
Analysis of the specific genes and laminae constructions of Retrosplenial cortex
*白坂 みさの(1)、花嶋 かりな(1)
1. 早稲田大学大学院先進理工学研究科
*Misano Shirasaka(1), Carina Hanashima(1)
1. Grad Sch Advanced Science and Engineering, Waseda University, Tokyo, Japan
Keyword: Retrosplenial cortex, spatial memory, circuit, dendritic bundle
The retrosplenial cortex (RSC), delineated as Brodmann’s areas 29 and 30, is a cortical area that contributes to a range of cognitive functions including contextual memory and spatial navigation. Neuroanatomical studies have characterized that RSC is interconnected with memory systems and sensory processing regions, suggesting that RSC is involved in spatial and non-spatial memory . While behavioral and functional studies of RSC have progressed over the last years, the underlying neural network and laminar assembly contributing to RSC function remain unknown. In this study, we identified “RSC-specific genes” which exhibit enhanced expression in the RSC in contrast to the neighboring cortices and examined the spatiotemporal dynamics of their expression along mouse development. In situ hybridization revealed changes in RSC-specific genes expression in subregions of RSC, retrosplenial granular cortex (RSG)and retrosplenial dysgranular cortex (RSD), from postnatal week 1-3. This results indicate that the RSC-specific genes may potentially contribute to alter the neural networks involved in spatial information processing. We further delivered GFP to RSC neurons generated at E15.5 by using in utero electroporation and examined dendritic bundles in layer 1 at the caudal RSG during postnatal development. Considering that the layer 1 of RSG receives inputs from CA1, alternative spatiotemporal expression of the RSC-specific genes during the postnatal period supports the idea that these genes may contribute to the construction of RSC-specific memory network. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-124
Signalling mechanisms underlying SSRI-induced adult neurogenesis in the mouse hippocampus
*樋口 裕城(1)、荒川 礼行(1)
1. 琉球大学大学院医学研究科
*Yuki Higuchi(1), Hiroyuki Arakawa(1)
1. School of Medicine, University of the Ryukyus, Okinawa, Japan
Keyword: adult neurogenesis, SSRI, hippocampus, RNA-seq
Adult neurogenesis occurs in limited brain regions, including the hippocampal dentate gyrus area and the subventricular zone in mouse models. The site-specific functions and underlying molecular mechanisms that execute neurogenesis in these brain regions remain unknown. There are several arguments in the function of adult neurogenesis relevant to stress-related mood disorders and antidepressant effects. Selective serotonin reuptake inhibitor (SSRI), a class of antidepressants, is known to activate adult neurogenesis in the dentate gyrus of the hippocampus. The facilitation of hippocampal neurogenesis induces neurotrophic mediatory processes and results in the replacement of existing neurons with new adult-born neurons to re-organize the hippocampal neural circuits. While these pharmacological processes appear in morphological levels, we still have major depression patients who do not respond to antidepressant treatment and thus resist to SSRI-induced neurogenesis. We hypothesize that signalling mechanisms underlying SSRI-activated pharmacological processes to facilitate neurogenesis may be responsible for that underlying treatment-resistant depression. In particular, we pay attention to an unknown function of microRNA, some of which are involved in the fluoxetine-induced neurogenesis in the hippocampus. We will bring fresh-baked data by investigating a long-term antidepressant treatment-induced neurogenesis in the mouse brain by a combination of behavioural, histological and RNA-sequencing analysis. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-125
小胞体膜分子 Derlin-1の成体海馬ニューロン新生における役割
An endoplasmic reticulum protein Derlin-1 maintains neural stem cell populations in the adult hippocampus
*村尾 直哉(1)、西頭 英起(1)
1. 宮崎大学医学部
*Naoya Murao(1), Hideki Nishitoh(1)
1. Dept of Med Sci, Univ of Miyazaki, Miyazaki, Japan
Keyword: Neural stem cell, Adult neurogenesis, Endoplasmic reticulum, Derlin
An endoplasmic reticulum (ER) has a quality control mechanism that ER resident stress sensors recognize unfolded or misfolded (malfolded) proteins and trigger the unfolded protein response (UPR). The UPR mediates the proper folding or degradation of malfolded proteins and the translational attenuation to inhibit the further production of ER proteins. A collapse of the ER quality control mechanism contributes to the onset and deterioration of several neurological disorders associated with impaired learning and memory. Adult neurogenesis is the process to generate new neurons from adult neural stem/precursor cells in restricted brain regions. Neurogenesis in the adult hippocampal dentate gyrus plays an important role in learning and memory formation, and its homeostasis is disrupted in several neurological disorders associated with memory impairment. ER quality control and adult neurogenesis are thought to be closely related to the mechanisms of learning and memory and neurological disorders, whereas the physiological relevance among them remains to be elucidated. An ER membrane protein Derlin-1 mediates ER-associated degradation of malfolded proteins (ERAD), and is known to be important for ER quality control mechanisms. In the present study, we investigated the function of Derlin-1 in neural stem cell-specific deficient (Derl1f/f; Nestin-Cre) mice in adult neurogenesis. We found that disruption of ER homeostatic maintenance mechanism of adult neural stem cells (aNSCs) caused abnormalities of adult neurogenesis and depletion of neural stem cell pool in Derl1f/f; Nestin-Cre mice. These results suggest that Derlin-1-mediated ER quality control in the central nervous system may play an important role in maintaining homeostasis of adult hippocampal neurogenesis. Furthermore, as a candidate factor causing depletion of stem cell pool due to deficiency of Derlin-1, we found that the protein expression of signal transducer and activator of transcription 5b (Stat5b) was decreased in Derlin-1-deficient aNSCs. In addition, disruption aNSC homeostasis due to Derlin-1 deficiency was rescued by Stat5b expression in the dentate gyrus. In this presentation, we will discuss the detail of homeostatic abnormalities of aNSCs and the mechanisms by which candidate factors such as Stat5b cause depletion of stem cell pool due to deficiency of Derlin-1. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-126
Transcriptional and post-transcriptional mechanisms of Brn2-mediated upper-layer neurogenesis in the cerebral cortex
*Zi Chao Ngiam(1), Carina Hanashima(1)
1. Waseda University
Keyword: Neurogenesis, Brn2, Neocortex, Foxg1
The 6-layered cytoarchitecture of the neocortex is evolutionarily conserved within the mammalian clade. Despite variations in their morphology, projection patterns, and molecular identity, lineage tracing studies show glutamatergic projection neurons in all layers are born from the same radial glial progenitor cell (RGC) population, with deep-layer (DL; Layer V and VI) neurons being produced first, followed by the upper-layer (UL; Layer II – IV) neurons, in an inside-to-outside lamination pattern. However, the mechanism underlying the reliable transition form DL to UL neurogenesis has yet to be fully elucidated. In this study we focus on Brn2, an UL neuron-specific transcription factor and its role in the Foxg1-expressing lineage. We first show that the change of the localisation of Brn2 from the cytosol to the nucleus within progenitors coincides with the time window of transition from DL to UL neurogenesis. However, gain-of-function studies demonstrate that Brn2 expression alone is insufficient to specify the UL neuronal fate and that it requires the presence of Foxg1, specifically at the time of UL fate determination to carry out its function. While the molecular mechanisms by which Foxg1 enables to respond to Brn2 requires further investigation, these results provide an important basis to the larger question of how progenitors maintain an invariant DL to UL neuron ratio during cortical neurogenesis. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-127
小脳グリア細胞に発現するMEIS1タンパク質の小脳発生期における機能解明
Function of the homeobox protein MEIS1 in cerebellar glial cells during cerebellar development
*足立 透真(1)、一條 研太郎(1)、大輪 智雄(1)、宮下 聡(2)、井上 由紀子(1)、井上 高良(1)、中村 卓郎(3)、星野 幹雄(1)
1. 国立精神・神経医療研究センター 、2. 新潟大学 脳研究所 基礎神経科学部門、3. 公益財団法人がん研究会 がん研究所
*Toma Adachi(1), Kentaro Ichijo(1), Tomoo Owa(1), Satoshi Miyashita(2), Yukiko Inoue(1), Takayoshi Inoue(1), Takuro Nakamura(3), Mikio Hoshino(1)
1. National center of Neurology and Psychiatry, 2. Brain Research Institute Niigata University, 3. Cancer Institute, Japanese Foundation for Cancer Research
Keyword: Homeobox protein, MEIS1, Bergmann glia, Cerebellar development
Myeloid ectopic viral integration site 1 homolog (Meis1) is a transcription factor and homeobox protein which is known to play a pivotal role in the development of the nervous system. It is known that disruption of Meis1 expression during development causes tumorigenesis such as neuroblastoma, therefore, understanding the function of MEIS1 during neuronal development is important in both fields of neurogenesis and neuro-oncology.In our previous study, we reported that Meis1 expressed in cerebellar granule progenitor cells (GCPs) plays an important role in the development of GCPs (Owa et al., Journal of Neuroscience, 2018). In order to elucidate the function of Meis1 in GCPs, we generated and analyzed Meis1-(flox/flox);Atoh1-Cre mouse, and found cerebellar atrophy, abnormal migration of GCPs, and defective formation of cerebellar granule cell (GCs) protrusions. However, GCPs are not the only cells expressing Meis1 in the developing cerebellum, and knockout of Meis1 in the whole cerebellum confirms a much more drastic phenotype than granule cell-specific knockout, suggesting that there is an additional important function of MEIS1 in the other cells during cerebellar development. In this study, we used single-cell RNA-seq data from the developing mouse cerebellum to show that Meis1 is also expressed in Bergmann's glia (BG), which is known to play an important role in proper differentiation and migration of GCPs. In addition, by knocking out Meis1 in multiple Cre driver mice, we found that Meis1 expression in BG is indispensable not only in BG itself but also in the development of GCPs and Purkinje cells, and its disruption causes drastic developmental defects. We are also investigating the signaling pathways downstream of MEIS1 expressed in BG which lead to the phenotypes we have observed. We would like to report on this as well. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-128
細胞内の一過的カルシウム濃度上昇は神経細胞移動の背景にある周期的な細胞形態形成を制御する
Calcium transients control a morphogenetic cycle underlying neuronal migratory movement
*堀金 慎一郎(1,2,3)、竹本―木村 さやか(1,2,3,4)、上條 諭志(3)、安達―森島 亜希(3)、藤井 哉(3)、尾藤 晴彦(3)
1. 名古屋大学環境医学研究所、2. 名古屋大学大学院医学系研究科、3. 東京大学大学院医学系研究科、4. JSTさきがけ
*Shin-ichiro Horigane(1,2,3), Sayaka Takemoto-Kimura(1,2,3,4), Satoshi kamijo(3), Aki Adachi-Morishima(3), Hajime Fujii(3), Haruhiko Bito(3)
1. Res Inst Envir Med, Nagoya Univ, Nagoya, Japan, 2. Grad Sch Med, Nagoya Univ, Nagoya, Japan, 3. Grad Sch Med, Univ of Tokyo, Tokyo, Japan, 4. PRESTO, Japan Science and Technology Agency, Saitama, Japan
Keyword: Neuronal migration, Ca2+ signaling, L-type VGCC
In spite of the critical importance of neuronal migration in the construction of brain architecture and neuronal circuits, morphogenetic rules operating neuronal migration during cortical layer formation have remained elusive. In particular, how numerous neurons can sequentially migrate in succession in a time-orchestrated manner within a limited space remains unsolved. We previously showed that migrating neurons responded to multiple extracellular factors that triggered Ca2+ influx via voltage gated Ca2+ channels (VGCCs), and further discovered a potential role for VGCC-driven spontaneous regenerative Ca2+ transients in neuronal migration. In keeping with this, we here found that radially migrating neurons in the cerebral cortex exhibited repeated spontaneous Ca2+ transients, while they underwent a characteristic, transient nuclear deformation or ‘rounding’. Furthermore, an evoked sustained Ca2+ elevation was able to trigger such nucleus deformation and maintained it throughout the duration of its transients. Intriguingly, the Ca2+ elevation was accompanied with multiple specific nucleus/cell morphology changes during a migratory movement: an initial acceleration followed by a halt in nucleus movement, a retraction in the trailing process, as well as a block in leading process extension. Thus Ca2+ elevation regulated three key morphogenetic components of neuronal migration. Mechanistically, Ca2+ influx via L-type VGCC was essential for nucleus rounding and nucleus movement. Consistently, expression of a dominant L-type VGCC gain-of-function mutation, associated with a syndromic autism spectrum disorder, induced an excessive nuclear rounding and perturbed cell migration. Together, our results shed light on the fundamental role of Ca2+ transients in orchestrating multistep morphogenetic cycles underlying neuronal radial migration. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-129
Nrf2陽性脳ペリサイトは幹細胞としての特性を獲得する
Nrf2-expressing pericytes acquire the traits of stemness
*佐久間 理香(1)、小林 未来(2)、小橋 瑞(2)、大西 真子(2)、前田 光代(3,4)、片岡 洋祐(3,4)、今岡 進(2)
1. 兵庫医科大学 解剖学細胞生物部門、2. 関西学院大学 生命環境学部、3. 理化学研究所 マルチモダル微細構造解析連携ユニット、4. 理化学研究所 細胞機能評価研究チーム
*Rika Sakuma(1), Miku Kobayashi(2), Rui Kobashi(2), Mako Onishi(2), Mitsuyo Maeda(3,4), Yosky Kataoka(3,4), Susumu Imaoka(2)
1. Department of Anatomy and Cell Biology, Hyogo College of Medicine, 2. Sch of Biol and Environmental Sci, Kwansei Gakuin Univ, Hyogo, Japan, 3. Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Hyogo, Japan, 4. Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research
Keyword: pericyte, Nrf2, Oxidative stress, stroke
Brain pericytes (PCs) are a mural support cell population elongated at intervals along the walls of capillaries. Recent studies reported that pericytes are multipotent cells that are activated in response to tissue injury and contribute to the regenerative process. Using a C.B-17 mouse model of ischemic stroke, it has been proposed that normal brain pericytes (nPCs) are converted to ischemic pericytes (iPCs), some of which function as multipotent stem cells. Furthermore, oxygen-glucose deprivation (OGD) promoted mesenchymal-epithelial transition in nPCs; however, nestin was not induced under OGD conditions (Neuroinflammation, 2016). In this study, we further investigated the mechanism of PC reprogramming phenomenon. In results, on day 3 after stroke, nestin+ iPCs within the ischemic areas co-expressed intercellular levels of reactive oxygen species (ROS) and the nuclear accumulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a key player in antioxidant defenses, but rarely in non-ischemic areas. Next, we isolated nPCs from the cortex of C.B-17 mice, and compared the traits of iPCs and nPCs, and the results obtained showed that nPCs and iPCs shared common pericytic marker mRNAs, including PDGFRβ, αSMA and NG2, and stem cell markers, nestin and Sox2 mRNAs were highly expressed in iPCs than nPCs. In addition, immunohistochemistry showed that the expression of Nrf2 was low in the cytoplasm of nPCs, but high in the nucleus and endoplasmic reticulum of iPCs, phosphorylated Nrf2 was only detected in iPCs, and downstream of Nrf2 significantly increased in iPCs than in nPCs. These results lead us to hypothesize that Nrf2 is required for the acquisition of stem cell activity following ischemia. We further examined that OGD/Reoxygenation and a treatment with tBHQ, a Nrf2 inducer, increased the expression of Nrf2 downstream and nestin in nPCs, and in Nrf2-overexpressing PCs, epithelial marker levels, including nestin, Sox2, and CDH1 (E-cadherin) mRNAs were elevated, which formed neurosphere-like cell clusters that differentiated into Tuj1-positive neurons. In conclusion, oxidative stress and Nrf2 are required for the generation of stem cells after stroke, and will contribute to the development of novel therapeutic strategies for ischemic stroke. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-130
脳梗塞病態における側脳室由来神経幹細胞と脳梗塞巣に出現する傷害誘導性幹細胞との関係に関する検討
Poststroke interaction between SVZ-derived NSPCs and regionally-induced iSCs within the ischemic area
*土居 亜紀子(1,2)、西江 英明(1)、澤野 俊憲(3)、中込 隆之(1,2)
1. 兵庫医科大学 先端医学研究所、2. 兵庫医科大学 先進脳治療学講座、3. 立命館大学 生命科学部
*Akiko Nakano-Doi(1,2), Hideaki Nishie(1), Toshinori Sawano(3), Takayuki Nakagomi(1,2)
1. Hyogo College of Medicine, Hyogo, Jpan, 2. Department of Therapeutic Progress in Brain Diseases, Hyogo, Japan, 3. Department of Biomedical Sciences, Ritsumeikan University, Shiga, Japan
Keyword: NSPCs, injury/ischemia-induced multipotent stem cells (iSCs)
Background: Neural stem/progenitor cells (NSPCs) mainly reside in specific neurogenic regions of the brain, such as the subventricular zone (SVZ). It is well documented that SVZ-derived NSPCs can migrate toward injured brain areas following ischemic stroke. We have previously demonstrated that cells with NSPC-like property, likely to originate from brain pericytes known as injury/ischemia-induced multipotent stem cells (iSCs), develop within ischemic areas and play a crucial role in neural regeneration. Purpose: Nevertheless, the relationship between SVZ-derived NSPCs and iSCs remains unexplored. To unveil this, the Cre-LoxP system was used to produce mice expressing yellow fluorescent protein (YFP) under the control of the Nestin promoter and studied the fate of NSPCs after ischemic stroke. Method: Nestin-CreERT2 Line4 mice [C57BL/6-Tg (Nes-cre/ERT2)4Imayo mice] were crossed with YFP reporter mice [B6; 129-Gt (ROSA) 26Sortm1 (EYFP)/J mice]. The Nestin-YFP mice (adult 8–16 week-old mice), thus obtained was subjected to focal cerebral ischemia by the middle cerebral artery occlusion method (MCA). Tamoxifen was administrated via a gastric tube in Nestin-YFP mice immediately after MCA occlusion to activate the Cre recombinase. Besides, brain sections were taken at 3 and 5 days poststroke and subjected to immunohistochemistry against YFP and a pericytic marker PDGFRβ. The latter is known to be expressed in iSCs. Result: YFP was selectively located within the SVZ under nonischemic conditions, but YFP+ cells were seen migrating toward the injured site after ischemic stroke from immunohistochemistry analysis. However, the migration capacity of SVZ-derived YFP+ cells was limited, and YFP+ cells were rarely observed in the ischemic cortex. Furthermore, while PDGFRβ+ cells were found within the ischemic region, they did not express YFP. Conclusions: These findings suggest that iSCs are not derived from NSPCs in the SVZ. Although we cannot completely rule out the possibility that NSPCs in the SVZ reached the ischemic region at the chronic phase, these results indicate that regionally-activated stem/progenitor cells—likely originating from brain pericytes rather than SVZ-derived NSPCs—acts like stem cells within the ischemic area at least during acute periods of stroke. However, future studies should clarify the precise roles of these two different stem cells in ischemic stroke. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-131
異なる系統に由来するNestin-GFP遺伝子改変マウスの表現型の比較解析
Analysis of the phenotypic differences between Nestin-GFP mice genetically derived from C57BL/6 and CB-17 strain mice
*西江 英明(1,4)、土居 亜紀子(1,2)、澤野 俊憲(3)、中込 隆之(1,2)
1. 兵庫医科大学先端医学研究所、2. 兵庫医科大学先進脳治療学講座、3. 立命館大学生命科学部、4. 日本臓器製薬株式会社
*Hideaki Nishie(1,4), Akiko Nakano-Doi(1,2), Toshinori Sawano(3), Takayuki Nakagomi(1,2)
1. Institute for Advanced Medical Sciences, Hyogo College of Medicine, 2. Department of Therapeutic Progress in Brain Diseases, Hyogo College of Medicine, 3. Department of Biomedical Sciences, Ritsumeikan University, 4. Nippon Zoki Pharmaceutical Co., Ltd.
Keyword: ischemic stroke, nestin, neural stem/progenitor cells, green fluorescent protein
Accumulating evidence reveals that endogenous neural stem/progenitor cells (NSPCs) are activated under pathological conditions, such as stroke. To elucidate the localization of these cells, researchers have created genetically modified mice wherein the neural stem cell marker, Nestin, is labeled with a green fluorescent protein (Nestin-GFP mice). Most genetically modified mice, including Nestin-GFP mice, have a genetic background (BG) of C57BL/6 strain mice that have several branches and collateral vessels in the middle cerebral artery (MCA), and the cerebral infarction model of these mice, by the occlusion of MCA, has the problem of significant variation in the infarct area across individuals and high mortality rates. However, CB-17 strain mice can evade these problems and exhibit a highly reproducible cerebral infarction following MCA occlusion (MCAO). Therefore, we created Nestin-GFP (CB-17 BG) mice by the consecutive backcrossing of Nestin-GFP (C57BL/6 BG) mice with CB-17 wild-type mice and compared their characteristics. The number of vessels branching from the MCA was significantly lower in Nestin-GFP (CB-17 BG) mice compared with Nestin-GFP (C57BL/6 BG) mice. The cerebral infarction volume following MCAO in Nestin-GFP (C57BL/6 BG) mice differed significantly across individuals, although Nestin-GFP (CB-17 BG) mice exhibited a minor variation in cerebral infarction volume across individuals. Furthermore, Nestin-GFP (C57BL/6 BG) mice occasionally died within a week after MCAO, whereas all Nestin-GFP (CB-17 BG) mice were alive for at least 28 days following MCAO. Immunohistochemical analysis of Nestin-GFP (CB-17 BG) mice revealed that GFP was expressed not only in the subventricular zone (SVZ), a well-known neurogenic region of the brain, but also in the infarct region. Neurospheres, a hallmark of NSPCs, were formed from both tissues after the isolation and culture of the SVZ and infarcted regions, respectively. They differentiated into various neural lineages, including neuronal cells, astrocytes, and oligodendrocytes. However, microarray analysis revealed that NSPCs from the SVZ and infarcted region had distinct characteristics, implying that these cells have a distinct origin. These results indicate that the localization of GFP-expressing NSPCs in Nestin-GFP (CB-17 BG) mice can be investigated for a long period and that these mice are effective as a tool to study the dynamics of NSPCs under pathological conditions, such as stroke. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-132
LDN193189はiSCを神経幹細胞様細胞へと誘導する
LDN193189 induces iSCs to neural stem cell-like cells
*湊 雄介(1)、土居 亜紀子(2)、大谷 佐知(3)、前田 誠司(1)、松山 知弘(4)、中込 隆之(2)、八木 秀司(1)
1. 兵庫医科大学解剖学細胞生物部門、2. 兵庫医科大学先端医学研究所、3. 兵庫医療大学共通教育センター、4. 兵庫医科大学先進脳治療学講座
*Yusuke Minato(1), Akiko Nakano-Doi(2), Sachi Kuwahara-Otani(3), Seishi Maeda(1), Tomohiro Matsuyama(4), Takayuki Nakagomi(2), Hideshi Yagi(1)
1. Dept Anat and Cell Biol, Hyogo Col of Med, Hyogo, Japan, 2. Inst Adv Med Sci, Hyogo Col of Med, Hyogo, Japan, 3. Gen Edu Ctr, Hyogo Univ of Health Sci, Hyogo, Japan, 4. Dept Therap Progress in Brain Deiseases, Hyogo Col of Med, Hyogo, Japan
Keyword: stem, BMP
Cerebral infarction causes serious sequelae so that an effective treatment is strongly desired. We have found ischemia-induced multipotent stem cells (iSCs) appeared in the infarct area of the post-stroke brain. Since iSCs can differentiate into various types of neuronal cells including neurons, they are expected to have clinical applications for cerebral infarction. However, iSCs rapidly lose their proliferation and differentiation potential through repeated passaging. At the 41st JNS meeting, we reported that high-density culture of iSCs could overcome the limitation of proliferative capacity, but not differentiation potential. In this report, we found that LDN193189, a BMP signaling inhibitor, overcomes the limitation of differentiation potential.
The cerebral infarcted area of mice caused by the middle cerebral artery occlusion was dissected and the primary culture of iSCs was prepared. The cells were divided into two groups at the first passage, LDN193189 was added to one of them, and high-density culture was performed. Analysis of gene expression profiles of iSCs showed that LDN193189 induced the expression of neural stem cell markers, while decreasing the expression of pericytes and mesenchymal markers. Thus we tried inducing neuronal differentiation and found that iSCs cultured in the presence of LDN193189 differentiated into Map2 and Tau-positive neurons. Furthermore, electrophysiological studies revealed that these differentiated neurons are electrophysiologically functional. iSCs cultured in the presence of LDN193189 could also be induced to differentiate into astrocytes.
These results indicate that LDN193189 overcomes the limitation of iSC differentiation potential and induces iSCs to become neural stem cell-like cells. We were able to develop an iSC culture method that preserves proliferative and neuronal differentiation potential by a combination of high-density culture and inhibition of BMP signaling. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-133
グリア細胞からニューロンへのダイレクトリプログラミングにおけるNeuroD1の発現量とリプログラミング効率
Expression level of the reprogramming factor NeuroD1 is critical for neuronal conversion efficiency from glial cells
*松田 花菜江(1)、松田 泰斗(1)、中島 欽一(1)
1. 九州大学
*Kanae Matsuda(1), Taito Matsuda(1), Kinichi Nakashima(1)
1. Kyushu University
Keyword: glial cells, reprogramming, neuron, conversion
Direct reprogramming is a technology to convert somatic cells from the original lineage to another by bypassing pluripotent state. We have previously shown that mouse microglia can be converted into neurons by the over expression of a single transcription factor, NeuroD1, through its pioneering activity. Several groups have reported in vivo neuronal reprogramming into neurons from glial cells, including astrocytes , which become reactive after brain damage and eventually contribute to glial scar formation. Although in vivo neuronal reprogramming from these two glial cells holds great promise as a therapeutic strategy, further improvement of neuronal reprogramming efficiency is warranted to supply enough new neurons for complete functional recovery from neurological injury and diseases. Although it has been assumed that neuronal reprogramming efficiency is attributable to expression levels of reprogramming factors (RFs), it has not been studied in detail how much it depends. Therefore, we here examined neuronal reprogramming efficacy from microglia and astrocytes under conditions of different expression levels of NeuroD1 in these two glial cell types. In contrast to the higher expression level, when we decreased the expression of NeuroD1, the neuronal conversion from microglia was dramatically diminished. On the other hand, increasing the NeuroD1 expression level by repeated lentiviral infections improved neuronal reprogramming efficiency from microglia and astrocytes. We also found that the combined expression of three RFs, Ascl1 and Brn2 together with NeuroD1, efficiently induced neuronal reprogramming, even when each expression level was low. In conclusion, our study offers efficient strategies to reprogram neurons from glial cells and will contribute to accelerating the development of therapeutic applications for brain injury and diseases. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-134
Setd8の発現低下はマウス海馬における加齢に伴う神経幹細胞の機能不全に関与している
Reduction of Setd8 expression is implicated in the age-related NSC dysfunction in the mouse hippocampus
*松原 周蔵(1)、松田 泰斗(1)、石龍 悠(1)、土井 浩義(1)、中川 拓海(1)、小田 尚伸(2)、中島 欽一(1)
1. 九州大学大学院医学研究院 基盤幹細胞学分野、2. 済生会熊本病院 総合腫瘍科 がんゲノムセンター
*Shuzo Matsubara(1), Taito Matsuda(1), Haruka Sekiryu(1), Hiroyoshi Doi(1), Takumi Nakagawa(1), Hisanobu Oda(2), Kinichi Nakashima(1)
1. Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 2. Saiseikai Kumamoto Hospital, Division of integrative medical oncology, Department of cancer genome medicine
Keyword: Adult neurogenesis, Neural stem cell, Aging, Epigenetics
Neural stem cells (NSCs) in the adult mouse hippocampus generate new neurons, which integrate into existing neural circuits and consequently support learning and memory. Hippocampal NSCs maintain their populations by controlling their activity to proliferate or to stay quiescent. However, NSCs gradually lose proliferative capability and fall into a deeper quiescent state during aging, reducing hippocampal neurogenesis associated with cognitive impairments. Recent studies have reported that alteration of microenvironmental niche affects NSC property, although cell-intrinsic mechanisms underlying the age-related property change of NSCs remain largely unknown. Here, we show that the reduction of Setd8, a sole enzyme that catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), underlies ever-changing age-related NSC property in hippocampus. To identify the factors associated with NSC dysfunction during aging process, we performed single-cell RNA-seq of EGFP-positive cells isolated from hippocampal dentate gyrus of Nestin-EGFP reporter mice at different time points (postnatal day 5, 12 weeks, and 24 weeks) and found that Setd8 was gradually downregulated with age. Conditional knockout of Setd8 in the adult NSCs decreased H4K20me1 levels, inducing deeper dormancy of NSCs accompanied by impaired neurogenesis in the hippocampus. We then performed RNA-seq analysis of intact and Setd8-knocked down NSCs in vitro. Up- or down-regulated genes by Setd8-knocked down displayed statistically significant overlaps with age-dependently up- or down-regulated genes in hippocampal NSCs, respectively. We also observed the argumentation of gene expression associated with NSC quiescence in Setd8-downregulated cells. Taken together, it is conceivable that the reduction of Setd8 expression accelerates age-dependent gene expression alteration associated with NSC quiescence and consequently induces deeper dormancy of NSCs, impairing neurogenesis in the hippocampus of aged mice. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-135
ヒト多能性幹細胞由来の視床下部・下垂体オルガノイドにおける下垂体幹/前駆細胞の探索
Isolation of pituitary stem/progenitor cells from human pluripotent stem cells-derived hypothalamic-pituitary organoid.
*野々山 葵(1,2)、河田 美穂(2)、小谷 侑(2)、亀山 俊樹(2)、齋藤 加奈子(2)、須賀 英隆(3)、長崎 弘(2)
1. 藤田医科大学大学院医学研究科、2. 藤田医科大学医学部生理学講座 I、3. 名古屋大学大学院医学系研究科糖尿病・内分泌内科学
*Aoi Nonoyama(1,2), Miho Kawata(2), Yu Kodani(2), Toshiki Kameyama(2), Kanako Saito(2), Hidetaka Suga(3), Hiroshi Nagasaki(2)
1. Grad Sch Med, Fujita Health University, Aichi, Japan, 2. Department of Physiology, School of Medicine, Fujita Health University, Aichi, Japan, 3. Department of Endocrinology and Diabetes, Grad Sch Med, Nagoya University, Aichi, Japan
Keyword: Pituitary, Stem/progenitor cell, Human iPSC
The pituitary gland is an endocrine organ that play an important role in homeostasis, such as growth, reproduction, metabolism, and stress response. And the number of each hormone-producing cell is regulated by life stage and environment. Recent studies in rodent showed that the existence of the stem/progenitor cells, which provide a lifelong supply of hormone-producing cells. However, the existence of human pituitary stem/progenitor cells remains unclear. In this study, we examined the human pituitary stem/progenitor cells using the hypothalamic-pituitary organoids derived from human induced pluripotent stem cells and embryonic stem cells. Fluorescence immunostaining identified pituitary stem/progenitor cell which express SOX2, CD9, PRRX1/2, PROP1, E-cadherin, Cytokeratin, S100β and FOXJ1 in the pituitary region of the human organoid. The cells in pituitary region also expressed EpCAM which we have identified as a specific surface marker for the anterior pituitary (unpublished data). The pituitary cells in the organoids were isolated by magnetically-activated cell sorting (MACS) using EpCAM. When EpCAM-positive cells were cultured in dish, we found some cells were adhesive, and some clustered in an island formation. After several passages, the clustered cells have lost and adhesive cells proliferated dominantly. The adhesive cells expressed pituitary stem/progenitor cell markers. In contrast, the mature pituitary cells expressing POMC were disappeared after the passages. Flow cytometry showed that about 98.5% of the adhesive cells expressed CD9, suggesting that the cells with a stem cell-nature dominantly existed after 6th passage. When the adhesive cells were differentiated with horse serum and FBS in 3D-culture system, some cells showed immuno-reactivity for adrenocorticotropic hormone or growth hormone. These results suggest that we have developed a method to isolate and expand pituitary stem/progenitor cell from human inducible stem cells. These results would contribute to both basic science and medicine, such as elucidating the regulatory mechanism of proliferation of hormone-producing cells in the anterior pituitary gland, and its application to regenerative medicine for pituitary defect after brain tumor. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-136
ヒトT細胞からの神経細胞への直接誘導と1細胞レベルでのキャラクタライゼーション
Generation and single-cell level characterization of neurons directly differentiated from human T lymphocytes
*斉藤 陽一(1)、石川 充(1)、Jonathan Moody(2)、Chung Chau Hon(2)、Jay Woo Shin(2)、赤松 和土(3)、岡野 栄之(1)
1. 慶應義塾大学医学部、2. 理研生命医科学研究センター、3. 順天堂大学医学部
*Yoichi Saito(1), Mitsuru Ishikawa(1), Jonathan Moody(2), Chung Chau Hon(2), Jay Woo Shin(2), Wado Akamatsu(3), Hideyuki Okano(1)
1. Keio Univ Sch Med, Tokyo, Japan, 2. RIKEN, IMS, Kanagawa, Japan, 3. Sch Med, Juntendo Univ, Tokyo, Japan
Keyword: Direct conversion, Single-cell RNA-seq, iN cell, Differentiation
Induced pluripotent stem cells have already proven to be a promising material for elucidating human neurological disease models, drug discovery and cell therapy for injury and deconditioning. However, preparing robust pathological models and resources for transplantation has been limited not only by the time required, but also by the labor and cost, as it requires first several weeks for complete reprogramming, then several months for quality control, and finally a long time for neuronal differentiation and maturation. On the other hand, induced neuronal cells (iN cells) programmed directly from human dermal fibroblasts may be very efficient, requiring only a few quick steps. However, obtaining human fibroblasts requires an invasive biopsy, which makes the research difficult. In this study, we focused on the direct conversion of T lymphocytes prepared from human adult peripheral blood mononuclear cells into properly functioning iN cells. While the gene set (NEUROD1/ASCL1/POU3F2/ZIC1) known to generate fibroblast-derived iN cells alone cannot generate large numbers of neurons, we succeeded in effectively producing target cells by simultaneously introducing Yamanaka 4 factors without the support of glial feeder cells. In fact, by mediating the transduction gene set with Sendai virus, an RNA virus with no risk of genomic integration, cells showing calcium activity were obtained within 3 weeks of culture. These cells exhibited stereotyped neuronal morphology and expressed several subtypes of neuronal markers. scRNA-Seq was used to perform comprehensive gene expression analysis to elucidate the details of the mechanism of iN cell generation. Furthermore, we applied RNA velocity analysis and trajectory analysis to estimate the cell dynamics. This study is not limited to understanding the mechanism of neuronal induction from various somatic cells. The more rapid and less invasive iN cell preparation method developed in this study can be applied as a resource for larger scale neurological disease modeling and cell therapy. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-137
Klf5遺伝子の胎児期神経幹細胞の細胞周期、分化、増殖への影響について
Klf5 gene effects on cell cycle, differentiation and proliferation of embryonic neural stem cells
*黒田 杏理(1)、林 義剛(1)、渕上 孝裕、北川 寛明(1)、依馬 正次(2)、等 誠司(1)
1. 滋賀医科大学生理学講座統合臓器生理学部門、2. 滋賀医科大学動物生命科学研究センター
*Anri Kuroda(1), Yoshitaka Hayashi(1), Takahiro Fuchigami, Hiroaki Kitagawa(1), Masatsugu Ema(2), Hitoshi Seiji(1)
1. Shiga University of Medical Science Integrative Phisioilogy, Shiga, Japan , 2. Shiga University of Medical Science Research Center for Animal Life Science, Shiga, Japan
Keyword: neural stem cell, cell cycle, Klf5
Krüppel-like factor (Klf) have 17 families in mammals which are orthologs of Drosophila melanogaster gene Krüppel. Klf family members are C2H2 zinc-fingers DNA-binding transcriptional factors and regulate various cellular function. Klf5 is one of the Klf members and plays a significant role in maintaining an undifferentiated state, cell cycle and differentiation of stem cells. However, the function of Klf5 in neural stem cells (NSCs) remain unknown. In this study, we investigate the distinctive function of Klf5 in the neural development. To investigate the effect of Klf5 overexpression on the self-renewal ability of NSCs, we performed a neurosphere assay using the ganglionic eminence of brains derived from E15.5 embryos of Klf5 overexpressing (OE) mice. The number of neurospheres from Klf5 OE brains was reduced as compared to that from wild-type controls, suggesting that Klf5 suppresses the self-renewal of NSCs. To reveal genes regulated by Klf5 , we performed RNA-seq analysis using neurospheres from Klf5 OE brain at E15.5. We found that the expression of the 886 and 615 genes was significantly upregulated and downregulated, respectively, in the neurospheres from Klf5 OE. Since gene ontology analysis of RNA-seq revealed that Klf5 OE is associated with the cell cycle and Notch pathway, we attempted to examine cell cycle time in detail using Neuro2a cells, a mouse neuroblastoma cell line, by measuring uptake dynamics of BrdU and EdU, which were separately added to the culture. The cell cycle length of the Klf5 OE Neuro2a cells was significantly decreased as compared to controls, suggesting that Klf5 OE induces rapid cell division. To investigate the effect of Klf5 on the development of brains, we performed qRT-PCR using E15.5 brains. The results showed significant upregulation of Hes1 and Nestin expression and tendency of downregulation of Neuron specific enolase, a neuronal marker, in the Klf5 OE brains compared to controls, suggesting that Klf5 OE expands the population size of neural stem/progenitor cells and suppress the neuronal differentiation. Thus, Klf5 contributes to the regulation of the cell cycle and the maintenance of stemness of neural stem cell. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-138
Ras-GAPs Control Neuronal Circuit Development in the Barrel Cortex Layer 4
*Madhura Subba Rao(1,2), Hiromi Mizuno(1,2), Takuya Sato(3), Takuji Iwasato(3,4), Hidenobu Mizuno(1,2)
1. Laboratory of Multi-dimensional Imaging, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto 860-0811, Japan, 2. Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan, 3. Laboratory of Mammalian Neural Circuits, National Institute of Genetics, Mishima 411-8540, Japan, 4. Department of Genetics, SOKENDAI, Mishima 411-8540, Japan
Keyword: Somatosensory barrel neonate cortex, Layer IV circuits, In-utero electroporation, Neurofibromatosis type 1
The cerebral cortex has a complex yet exquisite network of neuronal circuits which is important for advanced brain functional and cognitive purposes. To explore the molecular mechanisms of neuronal circuit formation, the tactile somatosensory pathway that connects the whiskers and cortex of rodents is useful. The rodent somatosensory barrel cortex layer 4 (L4) comprises a unique ‘barrel map’ that corresponds to facial whisker patterns. The whisker inputs via the thalamus form the neuronal circuit in the barrel cortex L4 during early postnatal development. Thalamocortical (TC) axon terminals are clustered in the barrels, ring-shaped distributions of neurons in the barrel cortex L4. Dendrites of L4 neurons are oriented toward the barrel center and receive inputs from the TC axon terminals. These traits make the barrel cortex a useful system to study neuronal circuit development. Our focus is Ras-GTPase activating protein (Ras-GAP) which possibly plays a role in the MAPK and PI-3 kinase pathways. We have created knockdown constructs for Ras-GAPs (NF1 and SynGAP) following which we transfected the constructs to the barrel cortex L4 using in-utero electroporation. We have analysed the effects of Ras-GAPs knockdown in the L4 circuit formation by the histological method. Our results suggest that Ras-GAPs knockdown L4 neurons may show reduced dendritic orientation. We also checked the colocalization of the post-synaptic L4 neuron dendritic spines and pre-synaptic TC axon terminals by confocal imaging. In the meeting, we would like to discuss the possible mechanisms for Ras-GAP-dependent L4 circuit formation. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-139
ティモシー症候群関連Cav1.2変異とDEE11関連Nav1.2変異はマウス大脳皮質において異なる様式で回路形成を障害する
Timothy Syndrome-associated Cav1.2 mutations and DEE11-associated Nav1.2 mutations impair neuronal circuit formation in different manners in mouse neocortex
*玉川（中川） 直(1,2)、菅生 厚太郎(3)、永田 英孝(3)、田川 義晃(1)
1. 鹿児島大学 大学院医歯学総合研究科、2. 理化学研究所 脳神経科学研究センター、3. 大日本住友製薬株式会社 基盤技術研究ユニット
*Nao Nakagawa Tamagawa(1,2), Kohtaroh Sugao(3), Hidetaka Nagata(3), Yoshiaki Tagawa(1)
1. Grad Sch Med Dent Sci, Kagoshima Univ, Japan, 2. RIKEN CBS, Japan, 3. Platform Technology Research Unit, Sumitomo Dainippon Pharma Co., Ltd., Japan
Keyword: Callosal projection, Neuronal migration, Neocortex development, Ion channel
The L-type calcium channel Cav1.2 and the voltage-gated sodium channel Nav1.2 regulate the intensity of neuronal activity and calcium signaling, and play important roles in the formation of neural circuits in the neocortex. Various mutations in both channels cause certain early-onset brain disorders that are considered to be associated with circuit malformations. Timothy syndrome (TS) is a multisystem disorder associated with cardiac and neurological symptoms, including long QT syndrome and autism spectrum disorder. Several Cav1.2 mutations have been reported as the cause of TS. Recently, a gain-of-function mutation, I1166T, was identified in patients with TS-like disorder. We performed in utero electroporation to express Cav1.2I1166T in layer 2/3 excitatory neurons of the primary somatosensory area in mice, and analyzed its effects on neuronal circuits. Impaired migration was seen in approximately 20% of Cav1.2I1166T-expressing neurons, and callosal projection was also markedly reduced. Inhibition of both Ca2+ influx and β-subunit interaction restored migration and projection. These results suggest that the I1166T affects both Ca2+ influx-dependent pathways downstream of Cav1.2 and β-subunit interaction, and impairs neural circuit formation [1]. The effects of G406R (the original mutation of TS) and other TS-associated mutations on neuronal circuits will also be shown in the presentation. Developmental and epileptic encephalopathy-11 (DEE11), another disorder we studied, is a neurological disorder characterized by the onset of seizures in early life and global developmental delay, usually accompanied by severe intellectual disability. DEE11 is caused by various mutations in Nav1.2. We introduced the Nav1.2 mutant into layer 2/3 excitatory neurons of mouse brain, and found that the mutation impairs migration and callosal projection, but in a manner different from Cav1.2 mutations. The contribution of Cav1.2 and Nav1.2 to the neuronal circuit formation may be different and will be discussed in the presentation.
[1] Tamagawa N.N., Kirino E., Sugao K., Nagata H., Tagawa Y. Involvement of Calcium-dependent Pathway and β subunit-interaction in Neuronal Migration and Callosal Projection deficits caused by the Cav1.2 I1166T Mutation in Developing Mouse Neocortex. Front Neurosci. 2021 Dec 8;15:747951. doi: 10.3389/fnins.2021.747951. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-140
Robo１シグナルを介したゴルジ体の動態変化は大脳皮質第2/3層錐体細胞の樹状突起 形態を決定する
Robo1 signaling-mediated Golgi apparatus dynamics determine the dendritic morphology of layers 2/3 pyramidal neurons in the cerebral cortex
*権田 裕子(1,3)、花嶋 かりな(2,3)
1. 東京医大・組織神経解剖、2. 早大・教育生物、3. 理研・CDB・大脳皮質発生
*Yuko Gonda(1,3), Carina Hanashima(2,3)
1. Dept. Histol. Neuroanat., Tokyo Med. Univ., Tokyo, Japan, 2. Lab. Dev. Biol., Waseda Univ., Tokyo, Japan, 3. Lab. Neocort. Dev., RIKEN CDB, Kobe, Japan
Keyword: dendrite, neocortex, excitatory neuron, Golgi apparatus
Neocortical neurons in each layer have layer-specific morphology and show specific gene expressions. Previous studies show that a strong interdependence exists between dendritic/ axonal morphology and information processing capabilities of a neuron. In particular, dendritic geometry strongly affects the action potential firing pattern of neurons, and is considered to be important for understanding brain function. Excitatory neurons, which are the major class of neocortical neurons, can be divided into two subtypes; pyramidal cells and spiny stellate cells, based on the morphology of apical dendrites. Pyramidal neurons are predominantly localized to layers 2/3, 5 and 6, and retain a single apical dendrite with multiple basal dendrites. In contrast, spiny stellate cells are mainly present in layer 4 and lack a prominent apical dendrite. However, the molecular mechanism by which layer-specific dendritic patterns is established remains largely unknown.
Here, we investigated the roles of Roundabout (Robo)1 signaling, one of the axon guidance molecules, in regulating dendritic morphology of upper-layer neocortical neurons. We previously reported that suppression of Robo1 expressed in neocortical layers 2/3 neurons causes extension of multiple apical neurites from the soma during the first postnatal week in mice. To investigate the intracellular mechanisms of Robo1-mediated dendrite development, we examined organelle dynamics during upper-layer neuron differentiation. We confirmed that the establishment of dendritic patterning in pyramidal neurons requires the regulation of multiple organelles in dendritic initiation. In addition, overexpression of Robo1 in layer 4 neurons that normally do not express Robo1 was accompanied by altered organelle localization, leading to the development of ectopic apical dendrites in these neurons. These studies demonstrate the existence of a molecular mechanism of upper neuron-specific dendrite formation via Robo1 signaling-mediated organelle dynamics in the neocortex. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-141
II型クラシックカドヘリンは精緻な神経網の配線過程で重複発現して初期回路基板の形成を担保する
Type II classic cadherins redundantly ensure the early circuit board formation for intricate neural network wiring
*井上 高良(1)、平賀 孔(1)
1. 国立精神・神経医療研究センター
*Takayoshi Inoue(1), Kou Hiraga(1)
1. National Center of Neurology and Psychiatry
Keyword: Cell Adhesion, Compartment, Wiring
Classic cadherins (Cdhs) are homophilic cell-cell, axon-axon, axon-axonic cell substrate and pre-post synaptic adhesion molecules. By generating CRISPR/Cas9-based multiple Cdh knockout mouse alleles, we have previously provided the first evidence that three type II Cdhs, Cdh6, Cdh8, and Cdh11, redundantly confer distinct adhesive codes to individual neuroepithelial cell at the embryonic day (E)8.5~9.5 to serve essential roles in the forebrain/midbrain compartmentalization and neurulation, both of which proceed under the robust control of the number, positioning, constriction, and fluidity of early neuroepithelial cells in the developing mouse neural plate/tube (Hiraga et al., Commun Biol 3, 574: 2020).
Here, we further determine physiological significance of Cdh6 and Cdh8 at the later stages of mouse neural development by making the best use of their CRISPR knockout alleles. As the results, we find out aberrant tract formation of the posterior commissure upon the disrupted forebrain/midbrain compartment boundary and branching defects of the trigeminal ophthalmic nerve in Cdh6/8 double knockout mice at E11.5 with 100% penetrance, while single knockout mice for Cdh6 or Cdh8 show few phenotypes along these nerve tracts. Given the overlapped expression of Cdh6 and Cdh8 in the corresponding neurons and/or axonic glial substrates for these nerve tracts at around E10.5, it is implicated that redundant type II Cdhs play indispensable roles in solidifying the early ‘circuit board’ for intricate neural network wiring. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-142
SIRPαTyr501のリン酸化・脱リン酸化と皮質錐体細胞の樹状突起伸長
Signal Regulatory Protein alpha (SIRPα) is involved in the dendritic growth of cortical pyramidal neurons through the phosphorylation and dephosphorylation of Tyr501 residue
*中村 史雄(1)、若槻 美祐(1)、瀧澤 光太郎(1)、實木・高橋 葵(1)
1. 東京女子医科大学
*Fumio Nakamura(1), Miyu Wakatsuki(1), Kohtaro Takizawa(1), Aoi Jitsuki-Takahashi(1)
1. Tokyo Women's Medical University
Keyword: dendrite, Sema3A, PTPdelta, SIRPalpha
Protein tyrosine phosphatase delta (PTPδ) is one of type IIa receptor type protein tyrosine phosphatases. We previously reported that PTPδ mediates Sema3A-induced dendritic arborization of cortical pyramidal neurons. However, its dephosphorylation target proteins are largely unknown. Phospho-proteomics analysis of PTPδ knockout and wildtype brains revealed that Signal Regulatory Protein alpha (SIRPα) was hyperphosphorylated at Tyr501 (Y501) residue in the knockout brains. Immunostaining of the brain sections with anti-phospho-Y501 SIRPα antibody revealed that olfactory bundle, cortical II/III layers, striate body, fimbria, corpus callosum, reticular thalamic nucleus, ethmoid thalamic nucleus, and pyramidal tract were hyperphosphorylated. We next examined whether SIRPα is involved in Sema3A-PTPδ signaling. Knockdown of SIRPα by siRNA transfection and overexpression of cytoplasmic deletion mutant of SIRPα suppressed Sema3A-induced growth cone collapse response of mouse dorsal root ganglion neurons. However, overexpression of non-phosphorylated mutant of SIRPα, SIRPα-Y501F, did not affect the collapse response. This suggests that phosphorylation/dephosphorylation of Y501 may not be involved in the signaling of Sema3A-repulsion. As hyperphosphorylation of SIRPα was observed in the cortical layers of PTPδ knockouts and poor arborization of basal dendrites of cortical pyramidal neurons in the knockouts, we examined the relation of SIRPα- phosphorylation and dendritic growth. Dendritic growth cones of primary cultured cortical pyramidal neurons showed transient dephosphorylation of the Y501 residue upon Sema3A-stimulation. Overexpression of SIRPα-Y501F mutant suppressed Sema3A-induced dendritic growth of primary cultured cortical neurons. In utero electroporation of SIRPα-Y501F to mouse brains showed that the apical dendrites of transfected cortical layer II/III pyramidal neurons were disoriented. These results indicate that the phosphorylation/dephosphorylation of SIRPα is involved in cortical dendritic growth. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-143
Sema3E-PlexinD1-RhoJシグナルによる嗅球新生ニューロンの樹状突起形成機構
Dendritic development of newborn neurons in the postnatal olfactory bulb is regulated by Sema3E-PlexinD1-RhoJ signaling
*澤田 雅人(1,2)、濱口 文人(1)、真野 尚道(1)、植村 明嘉(3)、澤本 和延(1,2)
1. 名古屋市大院医・脳研・神経発達・再生医学、2. 生理研・神経発達・再生機構、3. 名古屋市大院医・網膜血管生物学
*Masato Sawada(1,2), Ayato Hamaguchi(1), Naomichi Mano(1), Akiyoshi Uemura(3), Kazunobu Sawamoto(1,2)
1. Dept Dev Regen Neurobiol, Inst Brain Sci, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan, 2. Div Neural Dev Regen, NIPS, Okazaki, Japan, 3. Dept Retinal Vasc Biol, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan
Keyword: Postnatal neurogenesis, newborn neurons, dendritic development, olfactory bulb
Dendritic development of newborn neurons is a crucial step for the establishment and maintenance of neuronal circuits in the postnatal brain. Granule cells in the postnatal olfactory bulb (OB) are an ideal model to study the mechanisms for dendritic development in vivo. Even in the postnatal brain, granule cells are generated from neural stem cells in the ventricular-subventricular zone lining the lateral walls of the lateral ventricles, and continuously supplied into the OB. After the termination of migration in the OB, newborn granule cells extend a long apical dendrite, which is rarely branched in the granule cell layer (GCL) but extensively ramifies in the external plexiform layer, in addition to short basal dendrites within the GCL. The molecular mechanisms that determine such dendritogenesis patterns of newborn granule cells are not fully understood. Here, we show that the Sema3E-PlexinD1 signaling, which has been previously reported to maintain immature morphology of newborn granule cells during their migration, also plays a role in the regulation of dendritic development following migration termination. Genetic ablation of Sema3E or PlexinD1 enhanced lateral branching of the proximal part of an apical dendrite in newborn granule cells, whereas PlexinD1 overexpression suppressed it in a Rho binding domain (RBD)-dependent manner. We also found that the small GTPase RhoJ, which directly binds to PlexinD1 RBD in vascular endothelial cells, is expressed in differentiating newborn granule cells and also involved in the suppression of their lateral branching of the proximal part of an apical dendrite. Together, these results suggest that the Sema3E-PlexinD1-RhoJ axis shapes the dendritic morphology of newborn neurons in the postnatal OB. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-144
マウス胎仔小脳におけるプルキンエ細胞クラスターの形成
Formation of Purkinje cell clusters in the murine embryonic cerebellum
*羅 媛君(1,3)、Khoa Anh Tran(4)、平田 たつみ(2)、杉原 泉(1)
1. 東京医科歯科大学、2. 国立遺伝学研究所、3. 日本学術振興会、4. ハーバード大学医学大学院
*Yuanjun Luo(1,3), Khoa Anh Tran(4), Tatsumi Hirata(2), Izumi Sugihara(1)
1. Tokyo Med and Dent Univ, Tokyo,Japan, 2. Nat Inst of Gene,Mishima, Japan, 3. Japan Soc for the Prom of Sci,Tokyo, Japan, 4. Har Med Sch,Boston, USA
Keyword: mouse, protocadherin 10, FoxP2, neurogenin 2
The adult cerebellar cortex is compartmentalized into multiple (more than 40) longitudinal-stripes. Each stripe has distinct afferent and efferent projection patterns, and contains a particular subset of Purkinje cells. Purkinje cells (PCs) that occupy a single stripe share the same generation timing (birthdate) and have the expression profile of several molecules in common, including aldolase C (=zebrin). Purkinje cells (PCs) are mostly born from embryonic day 10.5 (E10.5) to E12.5. Our previous studies distinguished nine clusters of different generation timing and molecular expression profile in the E14.5 cerebellum, which divide and migrate into adult-type longitudinal stripes in the later period. However, the earlier processes of forming nine clusters before E14.5 are yet to be clarified. In this study, we analyzed and compared the spatial arrangement of PC subsets in E13.5 (and earlier) with E14.5 cerebellums. Purkinje cells were labeled according to the generation timing (E10.5, E11.5, and E12.5) using the neurogenin 2-CreER neurogenic tagging mouse strain (G2A mice). Serial coronal, sagittal, and horizontal sections were cut, and expression of marker molecules (FoxP2 and protocadherin 10) were also labeled by immunostaining. Digital images of serial sections were reconstructed into 3D models of PC clusters for the analysis.
In the E13.5 cerebellum, late-born PCs were located in the ventral part near the ventricular zone, and early-born PCs were located in the dorsal part near the dorsal surface of the cerebellum and far to the ventricular zone. Beside this dorso-ventral gradient of the distribution of early and late-born PCs, no clusters were observed at E13.5. In addition, the protocadherin 10 expression which characterizes the “ml” cluster in the E14.5 cerebellum, was observed mainly in the central cerebellum immediately dorsal to the area occupied by Purkinje cells. As a whole, the nine-cluster organization is scarcely formed in the E13.5 cerebellum, indicating a dynamic cluster formation process in the period between E13.5 and E14.5 in the developing cerebellum. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-145
脳に豊富に含まれているオレオイルリゾフォスファチジルエタノールアミンは大脳皮質初代培養神経細胞において神経突起伸長作用とグルタミン酸興奮性毒性にたいする保護作用を発揮する
Abundant oleoyl-lysophosphatidylethanolamine in brain exerts stimulation of neurite outgrowth and protection against glutamate toxicity in cultured cortical neurons
*近江 ウィリアム葵(1,2)、久野 和俊(2,3)、吉田 紘規(1,2)、川瀬 詩織(4)、三村 哲彦(2,3)、牧山 文亮(2,3)、中田 凌也(1,2)、羽二生 久夫(1,2,3)、塚原 完(5)、栗原 大河(6)、松田 佳和(7)、齋藤 直人(1,2,3)、植村 健(1,2,3,4)
1. 信州大・院総合理工、2. 信州大・先鋭領域融合研究群・バイオメディカル研、3. 信州大・院総合医理工、4. 信州大・基盤研究支援セ・遺伝子実験支援、5. 長崎大・院医歯薬学総合研究科・創薬薬理学、6. 日本薬科大・生命科薬学、7. 日本薬科大・臨床薬学
*William Aoi Omi(1,2), Kazutoshi Hisano(2,3), Hironori Yoshida(1,2), Shiori Kawase(4), Tetsuhiko Mimura(2,3), Fumiaki Makiyama(2,3), Ryoya Nakada(1,2), Hisao Haniu(1,2,3), Tamotsu Tsukahara(5), Taiga Kurihara(6), Yoshikazu Matsuda(7), Naoto Saito(1,2,3), Takeshi Uemura(1,2,3,4)
1. Grad. Sch. of. Sci. & Tech., Shinshu Univ., Nagano, Japan, 2. IBS-ICCER, Shinshu Univ, Nagano, Japan, 3. Grad. Sch. of. Med., Sci. & Tech., Shinshu Univ., Nagano, Japan, 4. Div. of Gene Res., Res. Cent. for Advanced Sci., Shinshu Univ, Nagano, Japan, 5. Dept. Pharmacol. Therap. Innov, Nagasaki Univ., Inst. Biomed. Sci., Nagasaki, Japan, 6. Div.Microbiol. & Mol. Cell. Biol. Nihon Pharmaceutical Univ, Saitama, Japan, 7. Div. Clinical Pharmacol., Nihon Pharmaceutical Univ, Saitama, Japan
Keyword: cultured cortical neuron, glutamate toxicity, mass spectrometry, neurite outgrowth
Lysophospholipids are bioactive phospholipids that have just one acyl chain and have been suggested to play an important role in physiological and pathophysiological processes. Lysophosphatidylethanolamine (LPE) is one of the lysophospholipids composed of a glycerol backbone with an ethanolamine head group, a phosphate group and a single fatty acid chain. Recently, using LPE prepared from egg yolk, we found that palmitoyl-LPE (16:0 LPE) and stearoyl-LPE (18:0 LPE) stimulate neurite outgrowth in cultured cortical neurons. Here, we analyzed the LPE composition in the mouse brain. Quantitative liquid chromatography–electrospray ionization tandem mass spectrometry analysis revealed that docosahexaenoyl-LPE (22:6 LPE) was most abundant in the 4-week-old mouse brain at approximately 4.2 nmol/mg weight of tissue, followed by docosatetraenoyl-LPE (22:4 LPE), oleoyl-LPE (18:1 LPE), arachidonoyl-LPE (20:4 LPE) at approximately 1.5, 1.5, and 1.2 nmol/mg tissue weight, respectively. Other LPE species were detected at lower concentrations. Several studies have shown that 18:1 LPE level was changed in several pathophysiological condition such as ischemia or Alzheimer’s disease model mice. However, the roles of 18:1 LPE in brain remain unknown. Therefore, in this study, we investigated the role of 18:1 LPE in central nervous system neurons. In cultured cortical neurons, we found that the application of 18:1 LPE stimulated neurite outgrowth. This effect was inhibited by Gq/11, phospholipase C inhibitor (PLC), protein kinase C (PKC), or mitogen-activated protein kinase (MAPK) inhibitor, suggesting that the action of 18:1 LPE on neurite outgrowth is mediated by mediated by Gq/11/PLC/PKC/MAPK pathway. We also analyzed the effect of 18:1 LPE on synapse formation. Moreover, we found that the application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. Collectively, our results demonstrate that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-146
Exploring the dendritic refinement dynamics of cortical neurons via in vivo high spatiotemporal-resolution imaging
*Luwei Wang(1,2), Shingo Nakazawa(1,3), Hidenobu Mizuno(4), Takuji Iwasato(1,2)
1. Laboratory of Mammalian Neural Circuits, National Institute of Genetics, Mishima, 2. Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, 3. Department of Basic Neurosciences, University of Geneva, Switzerland, 4. International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan
Keyword: dendritic refinement, in vivo imaging, barrel cortex
The specific arborization of dendrites determines the inputs the neuron receives, and the accurate dendritic pattern is formed and refined during development in an activity-dependent manner. It is critical to understand how dendrites refine their morphologies during development. To investigate the dendritic refinement dynamics of cortical neurons at early postnatal stages, our lab has used two-photon in vivo time-lapse imaging approaches (Mizuno et al., Neuron 2014; Nakazawa et al., Nature Commun. 2018). However, many details regarding the precise refinement features of cortical neuron dendrites are still largely unexplored because spatio-temporal resolution of previous studies was not sufficiently high. In this study, we first improved the spatial resolution of dendrite morphology imaging by using a membrane-bound RFP instead of a regular RFP. A regular RFP was strongly expressed in the cell body, which often disturbed the in vivo observation of precise morphology of dendrites, in particular that of proximal dendrites. In contrast, neurons labeled with the membrane-bound RFP, which are primarily localized on the cell membranes, visualized the precise dendritic morphologies. To improve the temporal resolution, we performed the imaging with 1-hour intervals instead of the previously performed 8-hour interval imaging, in which many refinement details were undetectable (Nakazawa et al., Nature Commun. 2018). We sparsely labeled barrel cortex L4 neurons with the membrane-bound RFP using the in utero electroporation-based Supernova method (Mizuno et al., 2014; Luo et al., Sci. Rep. 2016). TCA-GFP mice (Mizuno et al., 2014) were used to enable in vivo visualization of the barrel map. Then, we imaged dendrites of the same neurons for 8 hours at postnatal day 4, which is in the middle of the dendritic refinement process. With the current 1-hour interval imaging, we were able to catch subtle changes of individual dendrites including those of short-lived dendritic trees and branches. Our imaging also detected several transient refinement features of L4 neuron dendrites. We will discuss more of our recent analysis in the presentation. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-147
Secreted phospholipase A2 group V expressed by radial glia modulates LysoPtdGlc/GPR55-mediated nociceptive axon guidance in spinal cord development
*Adam T. Guy(1,2), Mariko Inoue(2), Yoshio Hirabayashi(3,4), Kei Hanafusa(4), Mitsuaki Yanagida(4), Kazuhisa Iwabuchi(4), Kei Yamamoto(5), Makoto Murakami(6), Hiroyuki Kamiguchi(2,7)
1. Grad Sch of Biostudies, Kyoto University, Kyoto, Japan, 2. Neural Cell Dynamics Lab, RIKEN CBS, Wako, Japan, 3. Cellular Informatics Lab, RIKEN CPR, Wako, Japan, 4. Institute for Environmental and Gender-Specific Medicine, Grad Sch of Medicine, Juntendo University, Tokyo, Japan, 5. Grad Sch of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Japan, 6. Center for Disease Biology and Integrative Medicine, Grad Sch of Medicine, University of Tokyo, Tokyo, Japan, 7. AMED-CREST, Japan Agency for Medical Research and Development, Tokyo, Japan
Keyword: AXON GUIDANCE, DEVELOPMENT, LIPID BIOLOGY
We have previously discovered that a novel lysophospholipid, lyso-phosphatidylglucoside (LysoPtdGlc), is a chemorepulsive axon guidance cue in developing spinal cord via its specific activation of GPR55 expressed by sensory neurons. LysoPtdGlc is a hydrolytic derivative of the membrane phospholipid phosphatidylglucoside (PtdGlc), which is produced by radial glia. Secreted phospholipase A2 (sPLA2) is a conserved family of enzymes that specifically cleave the sn-2-bound fatty acid chain of membrane phospholipids to produce diffusible lysophospholipids. We hypothesised that specific sPLA2 enzymes hydrolyse PtdGlc to LysoPtdGlc during LysoPtdGlc/GPR55-mediated axon guidance. We examined the mRNA expression of sPLA2 enzymes by radial glia and neurons cultured from embryonic mouse spinal cord using digital PCR, and found that radial glia specifically express sPLA2 V compared to non-glial spinal cord cells that expressed sPLA2 IB, III and X, but not V. Knockout of Pla2g5, the gene encoding sPLA2 V, induced statistically significant nociceptive axon projection errors in the spinal cord in vivo when compared to wildtype axon tracts (P <0.001, Wilcoxon’s matched-pairs analysis). We then developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol to directly measure the concentration of LysoPtdGlc in embryonic spinal cord of wildtype and Pla2g5 knockout mice. We conclude that the projection errors observed are caused by the inability of radial glia to hydrolyse PtdGlc into LysoPtdGlc. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-148
損傷後運動神経細胞軸索起始部のミクログリアおよびミトコンドリア分布の形態解析
Morphological analyses for mitochondrial localization in the axon initial segments (AIS) of nerve injured-motor neuron and microglial activation around AIS
*玉田 宏美(1)、桐生 寿美子(1)、沢田 蒼梧(1)、木山 博資(1)
1. 名古屋大学大学院
*Hiromi Tamada(1), Sumiko Kiryu-Seo(1), Sohgo Sawada(1), Hiroshi Kiyama(1)
1. Nagoya University
Keyword: axon initial segment, mitochondria, microglia, Focused Ion Beam / Scanning Electron Microscopy
The axon initial segment (AIS), which locates between the axon hillock and the beginning of myeline sheaths, is structurally and functionally characteristic. In response to axon injury, the appropriate disassembly of AIS occurs; however, the details of internal and external environmental changes seen in AIS after axon injury have not well characterized. In this study, we first focused on the mitochondrial localization in AIS, because the sufficient transportation of mitochondria from cell bodies to injured cites could be critical for proper regeneration after axon injury. Second, the activation and localization of microglia around AIS was analyzed. Axon injury induces the microglial activation and the activated microglia adhere to cell body; however, the microglial behavior to AIS is unknown. To address mitochondria and microglia behaviors after axotomy, a Focused Ion Beam/ Scanning Electron Microscopy (FIB/SEM) was used. FIB/SEM analysis allows to identify individual mitochondrion along the whole AIS length. Very few mitochondria were observed in the normal AIS, although abundant mitochondria were surprisingly located in the AIS after injury. The finding suggests that the mitochondrial sorting system in AIS is drastically alternated allowing the mitochondria influx to axons after injury. As for microglia, FIB/SEM showed that they adhered directly to AIS membrane along the whole length without insertion of any other elements. Some microglia whose processes were attached to AIS were also adhering to other distinct nerve-injured motor neuronal cell body. This suggests that the microglia could not distinguish between somatic and AIS membrane after axon injury. Because membrane-associated proteins of the AIS would be unique as potential generation site, they would lose their features and be shifted to the similar membrane composition to that of cell bodies after injury. In conclusion, the precise ultrastructural analysis with FIB/SEM revealed that axon injury caused the drastic changes of AIS in both the extracellular and intracellular milieu, which would be crucial responses for injured neuron to survive and regenerate. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-149
大脳皮質の発生過程における細胞の興奮性と脳梁形成
Cellular excitability and formation of the corpus callosum during development of the cerebral cortex
*大田 麗(1)、岡田 誠剛(2)、森 徹自(1)
1. 鳥取大学大学院医学系研究科保健学科生体制御、2. 倉敷芸術科学大学生命科学部
*Rei Ota(1), Masayoshi Okada(2), Tetsuji Mori(1)
1. Department of Biological Regulation, School of Health Science, Faculty of Medicine, Tottori University, Yonago City, Tottori, Japan, 2. Department of Medical Life Science, College of Life Science, Kurashiki University of Science and the Arts,Kurashiki, Okayama, Japan
Keyword: corpus callosum, pioneer neuron, development, excitability
Axons of pioneer neurons play important roles to establish correct neuronal circuits. The corpus callosum contains axons connecting the left and right cerebral hemispheres, and axons of callosal projection neurons are guided by axons of the pioneer neurons. The pioneer neurons locate in the cingulate cortex are born at embryonic day (E)13 in mice and their axons cross the midline at E15 in mice. During the central nervous system development, cellular activity is tightly regulated. And it has been shown that cellular activity affects not only differentiation/maturation of neural progenitors and immature neurons, but also axonal growth/arborization of postmitotic neurons. In this study, we investigated the role of cellular activity of neural progenitors producing the pioneer neurons and postmitotic pioneer neurons in formation of the corpus callosum. To inactivate or activate the neuronal excitability, a strongly inward rectifying potassium channel (Kir2.1) or a voltage-gated sodium channel (NaChBac) were overexpressed by in utero electroporation at E13 respectively. Moreover, we targeted different cell types by using two promoters: the CAG promoter and the synapsin 1 promoter. The CAG promoter is a ubiquitous promoter, and it is active in the progenitors and postmitotic neurons. On the other hand, the synapsin 1 promoter is a neuron specific promoter, and it is active in postmitotic neurons extending axons. First, Kir2.1 or NaChBac were overexpressed in the neural progenitors of the cingulate cortex by using the CAG promoter at E13. Introducing a CAG-Kir2.1 plasmid into the neural progenitors led to disruption of the laminar position and axonal projection of E13-born neurons. Second, we overexpressed Kir2.1 or NaChBac in the postmitotic pioneer neurons of the cingulate cortex by using the synapsin 1 promoter to disrupt axonal growth/arborization. From these experiments, we will discuss the morphological change of the pioneer neurons in the cingulate cortex and the formation of the corpus callosum as consequences of genetical manipulation of cellular activity during development. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-150
発達期のマウス大脳皮質におけるDEAF1遺伝子の異常が皮質ニューロンに与える影響
Effect of DEAF1 gene abnormalities on cortical neurons in mouse brain development
*西條 琢真(1)、永田 浩一(1)
1. 愛知県医療療育総合センター 発達障害研究所 分子病態研究部
*Takuma Nishijo(1), Koh-ichi Nagata(1)
1. Institute for Developmental Research, Aichi Developmental Disability Center, Aichi, Japan
Keyword: neurogenesis, DEAF1, in utero electroporation, wholl-cell patch clamp
The DEAF1 (Deformed Epidermal Autoregulatory Factor 1) gene, encoding a transcription factor, has been shown to be a possible responsible gene for autism spectrum disorders, intellectual disability and other neurodevelopmental disorders. However, pathophysiological mechanism caused by DEAF1 gene abnormalities in the cortical development is not cleared. We here analyzed the role of DEAF1 in the development of mouse cerebral cortex using in utero electroporation and whole-cell patch clamp technique. Acute knockdown of DEAF1 with the in utero electroporation technique in embryonic day 14 mouse cortical layer II/III pyramidal neurons caused suppression of dendritic arborization and affected dendritic spine morphology in cortical layer II/III pyramidal neurons during brain development. On the other hand, neuronal migration and axon elongation were not changed. These phenotypes were rescued by an RNAi-resistant version of DEAF1. Electrophysiological analyses using the whole-cell patch-clamp technique in postnatal day 7 to 10 mouse cortical layer II/III pyramidal neurons showed that DEAF1 knockdown reduced firing rate, excitatory synaptic transmission and inhibitory synaptic transmission. These results strongly suggest that DEAF1 plays an essential role in corticogenesis especially dendritic morphology and synaptic transmission. Its functional defects cause structural and functional impairment of cortical neurons, which is related to the pathophysiology of neurodevelopmental disorders. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-151
神経細胞に発現するGタンパク質共役型受容体GPR3は神経突起伸長、極性形成、細胞生存、軸索再生など多様な機能に関与する
Divergent functions of G protein-coupled receptor 3 in neurite outgrowth, polarity formation, survival, and axonal regeneration in neurons
*田中 茂(1)、益田 俊(1,2)、嶋田 直人(1)、白榊 紘子(1)、宮城 達博(1)、原田 佳奈(1)、秀 和泉(1)、酒井 規雄(1)
1. 広島大学大学院医系科学研究科 神経薬理学、2. 広島大学大学院医系科学研究科 視覚病態学
*Shigeru Tanaka(1), Shun Masuda(1,2), Naoto Shimada(1), Hiroko Shiraki(1), Tatsuhiro Miyagi(1), Kana Harada(1), Izumi Hide(1), Norio Sakai(1)
1. Dept Mol & Pharma Neurosci, Grad Sch Med, Univ of Hiroshima, Hiroshima, Japan, 2. Dept ophthal, Grad Sch Med, Univ of Hiroshima, Hiroshima, Japan
Keyword: Axonal regeneration, GPCR, cAMP, Neuronal survival
G protein-coupled receptor 3 (GPR3) is a member of the class A rhodopsin-type G protein-coupled receptors family that is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate Gαs protein without the interactions of ligands, thereby elevating the basal levels of intracellular cAMP. GPR3 levels in cerebellar granule neurons (CGNs) increase during neuronal development. Moreover, GPR3 has functions in neurite outgrowth and neuronal differentiation. We recently demonstrated that GPR3 stimulated neurite outgrowth in CGNs modulated via protein kinase A-, extracellular signal-regulated kinases-, and phosphatidylinositol 3-kinase (PI3K)-mediated signaling pathways. Additionally, GPR3-mediated activation of neurite outgrowth is associated with G protein-coupled receptor kinase-2 (GRK2)-mediated signaling and phosphorylation of the C-terminal serine/threonine residues of GPR3, which affects downstream PI3K-Akt signaling. Besides, GPR3 is expressed early in the development of hippocampal neurons and it is enriched in the longest tips of the neurites, accelerating neuronal polarization in a PI3K-dependent manner. Since GPR3 expression is sustained throughout life, we further investigated its possible involvement in neuronal survival and axonal regeneration based on retinal ischemia and optic nerve crush model in mouse retina. GPR3 knockout mice showed vulnerability of retinal ganglion neurons (RGNs) during aging and under ischemic conditions. In GPR3 knockout mice, GPR3 expression in RGNs affects axonal regeneration after optic nerve crush under zymosan-stimulated conditions. Axon regeneration was stimulated by GPR3 upregulation in RGNs, and this was further augmented with zymosan treatment. Taken together, these results indicate that GPR3 has functions in neurite outgrowth, polarity formation, survival and axonal regeneration in neurons, and the associated neuronal homeostasis throughout life. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-152
チロシンフォスファターゼとヘパラン硫酸プロテオグリカンを介した軸索側枝形成の分子機構
Molecular mechanism of protein tyrosine phosphatase and heparan sulfate proteoglycan-mediated axon collateral formation
*安村 美里(1)、猪口 徳一(1,2)、佐藤 真(1,3)
1. 大阪大学大学院医学系研究科、2. 福井医療大学看護学科、3. 大阪大学大学院連合小児発達学研究科
*Misato Yasumura(1), Tokuichi Iguchi(1,2), Makoto Sato(1,3)
1. Grad Sch Med, Osaka University, Osaka, Japan, 2. Dept Nursing, Fac Health Sci, Fukui Health Sci Univ, Fukui, Japan, 3. Div Dev Neurosci, United Grad Sch Child Dev, Osaka Univ, Osaka, Japan
Keyword: axon collateral formation, receptor protein tyrosine phosphatase, heparan sulfate proteoglycan
The corticospinal tract is a pathway that carry movement-related information from the cerebral cortex to the spinal cord. The corticospinal neurons project to various subcortical targets including the basilar pons via axon collateral branches. Developmental profile of the axon collateral formation to the basilar pons has been intensively investigated, however, the detailed molecular mechanisms underlying the axon collateral formation are still elusive. Recently, we found that knockdown of Ptprf encoding the receptor protein tyrosine phosphatase LAR (leukocyte common antigen-related protein) in the corticospinal neurons significantly suppressed the number and length of collaterals formed to the basilar pons. In the present study, to elucidate how LAR regulates the axon collateralization, we screened molecules interacting with the extracellular domain of LAR using affinity chromatography, and identified some heparan sulfate proteoglycans (HSPGs). We found that LAR directly bound to HSPGs by pull-down assays. To examine the involvement of HSPG in axon collateralization, we manipulated the expression of Ext1 and Ext2 genes encoding heparan sulfate biosynthesis enzymes, and analyzed collaterals formed to the basilar pons. We knocked out Ext1 and knocked down Ext2 in the corticospinal neurons by in utero electroporation of Ext1flox/flox:Fezf2-EGFP mouse cerebral cortex using Cre recombinase expression plasmid, Cre-dependent reporter expression plasmid, and shRNA plasmid at embryonic day 12.5. We also knocked out Ext1 in the neuron of the pons by tamoxifen administration to a Ext1flox/flox:Plxnd1-CreERT2:Fezf2-EGFP:Ai14 pregnant dam. Our preliminary data demonstrated that the manipulation of Ext1 and Ext2 gene expression might affect axon collateralization. In the meeting, we will show the results of ongoing analyses. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-153
Sema6A-PlxnA2/A4 signalingを介した嗅覚中枢神経回路の投射制御
Sema6A-PlxnA2/A4 signaling regulate the projection and collateral branch formation of olfactory bulb axons
*川崎 能彦(1)、平田 たつみ(1)
1. 国立遺伝学研究所
*Takahiko Kawasaki(1), Tatsumi Hirata(1)
1. National institute of genetics
Keyword: axon guidance
Neurons project their axonal branches to multiple targets in order to form neural circuits responsible for the integration and diffusion of information. In the case of olfactory bulb projection, collateral branches arising from the main shaft of the olfactory bulb axons project to a wide range of central regions, forming functional central olfactory circuits. Although collateral branches are involved in the formation of various neural circuits in the brain, it remains unclear how collateral branch formation is regulated in vivo.
In mice lacking Sema6A-PlxnA2/A4 signaling, the main shafts of olfactory bulb axons ectopically elongate to the caudal region of the telencephalon, deviating from its original projection pathway. On further analysis, we found that in mice lacking Sema6A-PlxnA2/A4 signaling, in addition to the ectopic elongation of the main shaft of olfactory bulb axons, following projection of the collateral branches to the cortical amygdala is markedly reduced. Interestingly, these abnormalities in the olfactory bulb projection were asymmetric between the left and right hemispheres. Although the mechanism causing this left-right asymmetry is not clear, the distance between the main shaft pathway and the cortical amygdala correlated with the presence or absence of collateral branch projections, suggesting that proximity to the cortical amygdala may induce collateral branches from the olfactory bulb axons. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-154
社会性ストレスによる視床シナプス結合の改編
Social stress-induced synapse remodeling in the somatosensory thalamus
*中山 寿子(1)、宮田 麻理子(1)
1. 東京女子医科大学
*Hisako Nakayama(1), Mariko Miyata(1)
1. Tokyo Women's Medical University
Keyword: synapse remodeling, stress, thalamus, corticosterone
It is not unusual for us in modern society that mental stress induces abnormality of sensation, including hyperesthesia and hypoesthesia. However, the underlying neuronal mechanisms are almost unknown. In mice, tactile information from whiskers is sent to VPM neurons in the thalamus through medial lemniscus fibers (MLFs). Most VPM neurons receive strong excitatory synaptic input from one MLF (mono-innervation) in mice after weaning age, while multiple MLFs (multiple-innervation) in the early postnatal stage. This mono-innervation is considered to be maintained throughout life so far. We investigated how social stress affects the mono-innervation at MLF-VPM synapses with electrophysiological and morphological methods. Mice were reared in group-housed or socially isolated conditions for four weeks after weaning of postnatal day 21. We found amplitude of EPSCs elicited by a single MLF was significantly reduced, and multiple-innervation at MLF-VPM synapses reappeared in socially isolated mice. Multiple-innervation but the reduction of EPSC amplitude was induced in group-housed mice administered corticosterone orally by mixing with the drinking water. In contrast, the reduction of EPSC amplitude but not multiple-innervation was observed in group-housed mice plucked bilateral whiskers every two to three days. We immunohistochemically revealed glucocorticoid receptors expressed in the thalamus, including the VPM. Furthermore, EPSC amplitude was significantly reduced, but multiple-innervation was not induced when social isolation started after sexual maturation (two-month-old). Our results showed that the MLF-VPM synapses are susceptible to social stress under isolated environments. It is suggested that social stress remodels the MLF-VPM synapses in two ways; one is a reduction of whisking activities that weaken MLF-EPSC amplitude, and the other is the activation of corticosterone-mediated signaling that allow innervation of additional MLFs. Taken social isolation after sexual maturation no longer induced multiple-innervation; the period from weaning to sexual maturation seems to be a critical period for MLF-VPM synapses to corticosterone signaling. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-155
視床体性感覚野におけるプレシナプスの機能的・形態的発達過程
Functional and morphological changes of the presynapse at the developing somatosensory thalamus
*緑川 光春(1)、宮田 麻理子(1)
1. 東京女子医科大学
*Mitsuharu Midorikawa(1), Mariko Miyata(1)
1. Tokyo Women's Medical University
Keyword: synapse, presynapse, synapse development
During the development, neural circuit change their function and morphology drastically before the maturation. However, these changes at the presynaptic side remains largely elusive at the most of the central nerve systems. Here, we focus onto the morphology of the terminal originated from the single axon to clarify the maturation of the neural circuit wiring. We utilized synapse at the rodent somatosensory thalamus. Somatosensory information of the whisker is conveyed to the ventral posteromedial thalamic nucleus (VPM). Previous studies including reports from our lab have shown that VPM relay neurons are innervated by multiple afferent fibers before the maturation, but redundant synapses are eliminated upon the developmental maturation and eventually dominated by a single fiber. Recently, we clarified the functional development of the single presynaptic terminal via direct patch-clamp recordings from the presynaptic terminals (Midorikawa & Miyata, 2021, PNAS). Using the same technique, we labeled the entire projection of the single afferent fiber by injecting neuro-tracer directly into the presynaptic terminal through the patch pipette. This method provided us the absolute conviction of the single fiber labeling. From the 3D confocal reconstruction, we examined the number, size and distribution of the terminals. We found drastic morphological changes of the presynaptic terminals during the development. With development, the number and the projection area of the terminals reduced drastically at the early stage of development, and the size of the terminals increased instead. The result suggests that smaller number of strong synapse is formed proximally to the single postsynaptic cell body after maturation, while abundant weak synapse is formed widely throughout the dendrites of single/multi postsynaptic cell(s). Combining these morphological data with functional data will lead us to understand the functional connective efficacy of the neural circuit. In addition, we will report the recent finding regarding the molecular bases of the functional development of the presynaptic neurotransmission ability. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-156
同期発火はシナプス結合形成を促進する
Synchronous firing promotes formation of synaptic connections
*鹿島 哲彦(1)、池谷 裕二(1,2,3)
1. 東京大・院薬・薬品作用、2. 東京大・Beyond AI研究推進機構、3. 脳情報通信融合研究セ
*Tetsuhiko Kashima(1), Yuji Ikegaya(1,2,3)
1. Lab Chem Pharmacol, Grad Sch Pharmaceut Sci, Univ Tokyo, Tokyo, Japan, 2. Inst AI Beyond, Univ Tokyo, Tokyo, Japan, 3. CiNet, Suita, Osaka
Keyword: Synaptic plasticity, Hebbian plasticity, Synchronous firing
During development of the central nervous system, synaptic connections are excessively generated and then reduced gradually through selective synaptic pruning. The candidate mechanisms of the selective pruning include the Hebb’s rule, which predicts that synaptic connections between neurons that fire synchronously survive developmental pruning. To experimentally verify this well-known rule, we induced synchronous firing in layer 2/3 neurons of the mouse somatosensory cortex that sparsely expressed channelrhodopsin 2 (ChR2) through in utero electroporation by non-invasive transcranial photostimulation method. We stimulated ChR2 on postnatal day 9-to-13 and examined synaptic connectivity using in vitro patch-clamp recordings from multiple layer 2/3 pyramidal neurons. The neocortex that received chronic photostimulation exhibited higher probabilities of synaptic connections between ChR2-positive neurons, compared to ChR2-negative neurons or ChR2-positive neurons in the non-stimulated neocortex. The photostimulation did not induced any alternations in either the basic electrophysiological properties or the morphological characteristics of the stimulated neurons, except that spines of their basal dendrites were enlarged. These results are consistent with the Hebb’s prediction, suggest that developmental synchronous firing is actively involved in the neural circuit formation in neocortex. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-157
発達期齧歯類の皮質-運動ニューロンシナプスにおけるシナプス除去
Elimination of cortico-motoneuronal synapses during development in rodents
*大野 孝恵(1)、福田 諭(1)、崎村 建司(2)、桜井 正樹(1)
1. 帝京大学医学部生理学講座、2. 新潟大学脳研究所
*Takae Ohno(1), Satoshi Fukuda(1), Kenji Sakimura(2), Masaki Sakurai(1)
1. Teikyo Univ.Sch.Med., 2. Brain Research Institute, Niigata Univ.
Keyword: cortico-motoneuronal synapses, GluN2B-containing NMDA receptors, synapse elimination, whole cell recordings
We previously found that corticospinal axons make monosynaptic connections with motoneurons in rodents of 1st to 2nd postnatal weeks, which had been believed to be present only in higher primates. Developmental time course of the synapse elimination studied by whole cell recordings of retrogradely labeled forearm motoneurons showed that the positive ratio of monosynaptic cortico-motoneuronal EPSCs decreased from P14 toward P21. The mechanisms of those synapse eliminations, however, are still unknown. Since the corticospinal synapse elimination from the ventral side occurred in a postsynaptic GluN2B-dependent manner according to our previous work of in vitro slice co-culture, we studied the involvement of postsynaptic GluN2B for this cortico-motoneuronal synapse elimination. Cortico-motoneuronal EPSCs were present in about 30 % of 2B-knocked out motoneurons, using Grin 2B-floxed mice expressing AAV-Cre selectively in spinal motoneurons, which suggests that this synapse elimination was at least in part dependent on postsynaptic GluN2B. We first studied the synaptic events underlying the corticomotoneuronal elimination by whole cell recordings using acute spinal slices. The second responses of paired pulse stimulation showed significant depression until P13 when the elimination begins, suggesting that presynaptic release is reduced. After P14, the ratio of depression decreased, which tend to approach one. There was a significant decline of depression ratio during the elimination phase. We examined the developmental change of miniature EPSC (mEPSC) amplitude, which partly reflect the postsynaptic alteration. Cortico-motoneuronal mEPSCs were recorded by inducing asynchronous quantal release by substitution of Sr2+ for Ca2+ in external solution. To observe the involvement of postsynaptic GluN2B on the synapse elimination, we next studied the developmental changes of synaptic transmission including AMPA/ NMDA ratio, GluN2B/ 2A ratio, and the amplitudes of mEPSCs in GluN2B knocked out motoneurons, which was achieved by crossing GluN2B-floxed mice and Cre-expressing mice.. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-158
ペリニューロナルネットリンクプロテインHapln4欠損マウスにおいてグリア細胞が発達期calyx-MNTBシナプス形成に与える影響
Assessment of glial cells on the development of calyx-MNTB synapse in the Hapln4-deficient mice
*大橋 俊孝(1)、谷 祐一(1)、宮﨑 晴子(1)、野島 弘二郎(1)、兼城 一媛乃(1)、堀 哲也(2)
1. 岡山大学、2. 沖縄科学技術大学院大学
*Toshitaka Oohashi(1), Yuichi Tani(1), Haruko Miyazaki(1), Kojiro Nojima(1), Himeno Kaneshiro(1), Tetsuya Hori(2)
1. Grad Sch Med Dent Pharm Sci, Okayama University, Okayama, Japan, 2. Okinawa Inst of Sci and Tech Grad, Okinawa, Japan
Keyword: Perineuronal net, proteoglycan, synapse, glial cell
The principal cells of the medial nucleus of trapezoid body (MNTB) receive their large single input, which is known as calyx of Held, from the ventral cochlea nucleus. MNTB neurons are enclosed in densely organized extracellular matrix structures, known as perineuronal nets (PNNs). PNNs are typically found around fast-spiking GABAergic interneurons expressing parvalbumin but interestingly also exist surrounding other neurons, such as the neurons in the MNTB. This characteristic localization prompted the hypothesis that PNNs might play a role in the maintenance and formation of large fast-signaling synapses. We have recently reported that hyaluronan and proteoglycan binding link protein 4 (Hapln4) is important for brevican localization specific at the perisynaptic space between the calyx of Held terminals and principal neurons (Nojima et al., Front Cell Dev Biol 2021). To further elucidate the functional role during postnatal development, we have investigated the electrophysiological and morphological properties of calyx-MNTB synapses in Hapln4-knockout (KO) mice. The results indicated that MNTB neurons are innervated by multiple synaptic terminals in Hapln4-KO, whereas the neurons receive large single input in wild-type (Okazaki et al. in JNS 2020, Kobe). We herein assessed the contributions of glial cells in synaptic pruning at postnatal days (P) 7 and 14, i.e., prior to and just after onset of hearing and PNN formation. Our results may provide a clue to understand the functional significance of Hapln4 in the calyx of Held synapse formation. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-159
Early postnatal developmental period for ASD-related social behavioral circuits
*Goichi Miyoshi(1)
1. Grad Sch Med, Gunma Univ, Japan
Keyword: Social behavior, Inhibitory circuit development, Early postnatal, Autism spectrum disorder (ASD)
Autism spectrum disorders (ASD) are characterized by social communication deficits and restricted/repetitive behavioral patterns (DSM-5), with a prevalence around 1 to 2% of all children (USA: CDC estimate). While the vast majority of ASDs are thought to emerge through a combination of genetic and environmental risk factors, a percentage (~5%) of ASD cases are syndromic, in that they are caused by highly penetrant mutations in a single gene, such as MECP2 (Rett or duplication), FMR1 (Fragile X) or SHANK3 (Phelan-McDermid). Despite their relative rarity, these syndromic forms of ASD have provided key insights into the broader etiological basis of ASD. Of particular interest, many syndromic ASD models show an increase in the cortical synaptic excitation-inhibition (E/I) ratio, a phenomenon that has been hypothesized to underlie the cognitive and behavioral symptoms of ASD. While abnormalities in GABAergic inhibitory circuits have been implicated in the etiology of ASD, when and how ASD-related social dysfunction emerges during development is poorly understood. Recently, we have selectively manipulated the expression of the syndromic ASD gene FOXG1 in rodent models to identify that the second postnatal week is the critical period to establish juvenile inhibitory circuits and to prevent ASD social phenotypes. We find that either increased or decreased FoxG1 levels result in social impairments but only when alterations take place in both excitatory and inhibitory populations, indicating a pre-and postsynaptic role for FoxG1 in GABAergic circuit development. While we found that a decrease in GABAergic tone exacerbates social impairments, supplementation of GABAergic interneuron precursors prior to the critical period ameliorates ASD-related circuit and behavioral phenotypes of the FOXG1 ASD model animals. Our results reveal the developmental timing and inhibitory circuit mechanisms that are promising for therapeutic intervention of ASD. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-160
脳幹NMDA受容体欠損マウスが明らかにする体性感覚神経回路の精緻化
Somatosensory neural circuit refinement revealed by brainstem NMDAR deficient mice
*木村-中嶋 ちえみ(1,2)、鈴木 亜友美(1,2)、岩里 琢治(1,2)
1. 国立遺伝学研究所 神経回路構築研究室、2. 総合研究大学院大学 生命科学研究科遺伝学専攻
*Chiemi Kimura-Nakajima(1,2), Ayumi Suzuki(1,2), Takuji Iwasato(1,2)
1. Laboratory of Mammalian Neural Circuits, National Institute of Genetics, Mishima, Shizuoka, Japan, 2. Department of Genetics, SOKENDAI, Mishima, Shizuoka, Japan
Keyword: whisker-barrel system, brainstem, NMDA receptors, conditional knockout mice
The rodent whisker-barrel system is an excellent model for studies of activity-dependent neural circuit formation, in which NMDA-type glutamate receptors (NMDARs) play a key role. In rodents, somatosensory information from the whiskers reaches through the brainstem and thalamus to the cortex layer 4, where there are whisker-related patterns called barrelettes, barreloids, and barrels, respectively. These patterns are generated from the periphery to the center during the first postnatal week in activity-dependent manners. To reveal the role of brainstem NMDARs in the somatosensory neural circuit refinement, we here generated the brainstem-specific NR1 knockout (Bs-NR1KO [K]) mice, which lack NR1, the essential NMDAR subunit, specifically in the brainstem. Barrelette patterns were absent in these mice, suggesting that the brainstem NMDAR is important for the barrelette formation. On the other hand, although barreloids and barrels were not normal, these abnormalities were weaker than we expected. In Bs-NR1KO [K] mice, the recombination ratio in the brainstem vPrV, where barrelettes are generated in wild-type mice, was not high. To increase the recombination ratio in the vPrV, we introduced another line of transgenic mice [H] that also expresses Cre recombinase in the vPrV into Bs-NR1KO [K] mice and obtained Bs-NR1KO [K&H] mice. All barrelettes, barreloids, and barrels were almost disappeared in Bs-NR1KO [K&H] mice, suggesting that the brainstem NMDARs are essential not only for barrelette formation but also for barreloid and barrel formation. Previous studies in our laboratory and others have focused on roles of molecules expressing in the thalamus and the cortex in the cortical circuit refinement. Here, we revealed a role of NMDARs in the brainstem, which does not project directly to the cortex, in the cortical neural circuit refinement. Besides, the result that barrels can be formed even without detectable barrelettes in Bs-NR1KO [K] mice was somewhat unexpected because barrels are thought to be formed by using barrelettes as a template. Detailed analysis of Bs-NR1KO [K] mice may provide new insights into some aspects of neural circuit refinement. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-161
自閉症モデルマーモセットの背内側前頭前皮質におけるシナプスのターンオーバーとシナプス間相互作用の解析
Synaptic turnover and inter-synapse interactions in the dorsomedial prefrontal cortex of autism model marmosets
*野口 潤(1)、渡邉 惠(1)、磯田 李紗(1)、中垣 慶子(1)、境 和久(1)、菅野 江里子(2)、冨田 浩史(2)、渡我部 昭哉(3)、山森 哲雄(3)、水上 浩明(4)、一戸 紀孝(1)
1. 国立精神・神経医療研究センター 神経研究所、2. 岩手大学 理工学部 、3. 理化学研究所 脳神経科学研究センター 、4. 自治医科大学 分子病態治療研究センター
*Jun NOGUCHI(1), Satoshi WATANABE(1), Risa ISODA(1), Keiko NAKAGAKI(1), Kazuhisa SAKAI(1), Eriko SUGANO(2), Hiroshi TOMITA(2), Akiya WATAKABE(3), Tetsuo YAMAMORI(3), Hiroaki MIZUKAMI(4), Noritaka ICHINOHE(1)
1. National Center of Neurology and Psychiatry, Tokyo, Japan, 2. Grad Course Biol Sci, Iwate University, Iwate, Japan, 3. Center Brain Sci, RIKEN, Saitama, Japan, 4. Div Genet Therapeutics, Jichi Med Univ, Tochigi, Japan
Keyword: dendritic spines, Two-photon microscopy, autism, marmoset
Coordinated synapse generation and pruning are mediated by interactions between synapses and are essential for proper refinement of the neuronal network. However, this process might be impaired in patients with autism spectrum disorder (ASD). We previously created an ASD model marmoset by orally administering valproic acid (VPA) to a pregnant marmoset. We then examined the affected offspring (VPA model marmosets). The VPA offspring showed abnormalities in third-party reciprocity and inequity aversion, reminiscent of impaired higher-order social skills. These measures have not been well examined in rodent ASD models to date.
To investigate synaptic changes underlying impaired social deficits, we evaluated the synaptic properties of the dorsomedial prefrontal cortex of the adult VPA offspring. This was done by directly observing postsynaptic dendritic spines and presynaptic boutons with two-photon microscopy. The same neocortical neurites were observed every three days, and the results indicated that there was an enhancement of spine and bouton generation and elimination in the autism / VPA model, suggesting excessive instability of the neuronal circuits.
Furthermore, we observed that the spines newly generated in the VPA model tended to be located in relatively close proximity; in other words, they had a greater tendency to cluster, which was reversed by nasal administration of oxytocin. On the other hand, by analyzing relative spine volumes in both VPA marmosets and control marmosets, we found that spine elimination was more pronounced in smaller spines in the model animals.
Finally, we applied microarray gene expression analysis to the social neocortex areas of marmosets and compared it with data from human ASD patients. The results showed that there were significant correlations between the fold change (logFC) values of gene expression in adult model marmosets and the logFC values of postmortem human ASD.
These results support the possibility that changes in synaptic stability and/or synaptic interactions could underlie the symptoms of autism. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-162
運動学習による大脳皮質運動野での入力依存的なシナプス可塑性
Presynaptic supervision of cortical synaptic plasticity in motor skill learning
*孫 在隣(1)、川口 泰雄(1,2,3)、窪田 芳之(1,2)
1. 生理学研究所、2. 総合研究大学院大学、3. 玉川大学 脳科学研究所
*Jaerin Sohn(1), Yasuo Kawaguchi(1,2,3), Yoshiyuki Kubota(1,2)
1. National Institute for Physiological Sciences (NIPS), Aichi, Japan, 2. Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Aichi, Japan, 3. Brain Science Institute, Tamagawa University, Tokyo, Japan
Keyword: motor learning, spine plasticity, motor cortex
Learning novel motor skills rewires neuronal connections by generating new synapses in the motor cortex. Of particular importance are the spines on pyramidal cell dendrites where synapses are formed and maintained during learning. However, little is known about the sources of synaptic inputs that are concomitantly rearranged. Here, we show that spine dynamics over the course of perfecting a motor skill depend on cortical and thalamic inputs. Post hoc characterization of the presynaptic axon terminals on new spines revealed that motor skill improvement coincided with formation of spines innervated by corticocortical axons more frequently than those innervated by thalamocortical axons. New thalamocortical synapses appeared less frequently, but survived longer and increased spine size more than new corticocortical synapses that were largely pruned. The input-dependent fate of learning-associated new spines suggests that pyramidal cell dendrites in motor cortex use a neural circuit division-of-labor during skill learning, with dynamic teaching contacts from top-down intracortical axons followed by synaptic memory formation driven by thalamic axons. Dual spine supervision may govern diverse skill learning in neocortex. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-163
大脳皮質錐体細胞におけるアグリカンの強制発現により異所的なペリニューロナルネットが形成される
Ectopic formation of perineuronal nets by overexpressing aggrecan in cortical pyramidal neurons
*宮田 真路(1)、芦野 颯斗(1)、武渕 明裕夢(1)
1. 東京農工大学農学府
*Shinji Miyata(1), Ryuto Ashino(1), Ayumu Mubuchi(1)
1. Grad Sch Agr, Tokyo Univ Agr and Tech
Keyword: Perineuronal nets, Critical period plasticity, Extracellular matrix, Aggrecan
Cortical plasticity is most evident during a critical period in early life. Maturation of a subset of inhibitory interneurons expressing calcium-binding protein parvalbumin (PV cells) has been proposed to play a crucial role in defining the timing of the critical period. During postnatal development, chondroitin sulfate proteoglycans (CSPGs) condense around the soma and proximal dendrites of PV cells, forming perineuronal nets (PNNs), a specialized extracellular matrix structure that interdigitates with synaptic contacts. PNNs are composed of several CSPGs and hyaluronan, and interaction between CSPGs and hyaluronan is stabilized by tenascin-R and cartilage link proteins. Enzymatic disruption of PNNs with chondroitinase ABC reactivates critical period plasticity in adult animals, indicating that PNN formation around PV cells is responsible for the closure of the critical period. However, the molecular mechanisms underlying the cell-type-specific formation of PNNs remain unknown. Here we hypothesized that ectopic expression of specific components of PNNs enables the formation of PNNs around non-PV cells, such as excitatory pyramidal neurons. We first searched components of PNNs that were selectively expressed by PV cells using a database of single-cell RNA sequencing and found that aggrecan, a large aggregating CSPG, was predominantly expressed by PV cells. To discriminate between exogenous and endogenous aggrecan, we constructed a plasmid vector expressing mouse aggrecan fused with green fluorescence protein (GFP) under the control of a chicken beta-actin promoter. To achieve pyramidal neuron-specific gene expression, we performed neocortex-directed in utero electroporation since pyramidal neurons are born in the embryonic cortical ventricular zones. GFP-fused aggrecan and cytosolic form of mCherry-expressing plasmid were introduced at embryonic day 12 or 14, and the brains were analyzed two months after birth. We found that forced expression of GFP-fused aggrecan resulted in ectopic emergence of PNN-like structures around pyramidal neurons. Transfected, but not the neighboring untransfected pyramidal neurons were surrounded by PNNs, indicating that overexpression of aggrecan cell-autonomously affects PNN formation. Our study may offer a novel approach to elucidate how PNNs control the critical period plasticity. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-164
生後早期のミダゾラム曝露はクロマチンのアクセス性を持続的に変化させ、成体ニュ―ロン新生および認知機能を低下させる
Early-life midazolam exposure persistently changes chromatin accessibility to impair adult hippocampal neurogenesis and cognition
*土井 浩義(1,2)、松田 泰斗(1)、山浦 健(2)、中島 欽一(1)
1. 九州大学医学研究院 基盤幹細胞学、2. 九州大学大学院医学研究院 麻酔・蘇生学
*Hiroyoshi Doi(1,2), Matsuda Taito(1), Ken Yamaura(2), Kinichi Nakashima(1)
1. Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 2. Department of Anesthesiology and Critical Care Medicine, Graduate school of Medical Sciences, Kyushu University
Keyword: Neurogenesis, Neural stem cell, Midazolam, Chromatin accessibility
Adult neural stem/progenitor cells (aNS/PCs) in the subgranular zone (SGZ) of the adult hippocampal dentate gyrus (DG) proliferate and give rise to new neurons continuously throughout life to maintain hippocampus-dependent cognitive functions. Recent studies have demonstrated that gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter in the adult brain, suppresses aNS/PC proliferation in the SGZ. Midazolam(MDZ), a GABAA receptor agonist, is widely used in clinical and surgical procedures, such as induction and maintenance of anesthesia, and sedation. Although previous studies indicated that early-life repeated (but not single) exposure to GABAA receptor agonists including midazolam c is associated with neurocognitive outcomes in later life, however, little is known how anesthesia leads to long-term CNS dysfunctions. In this study, we investigated whether early-life exposure to MDZ affects NS/PC behavior and consequently impairs cognitive deficits in adulthood. Here we show that early-life exposure to midazolam (MDZ), a widely used drug in pediatric anesthesia, persistently alters chromatin accessibility and expression of quiescence-associated genes such as Notch2 and REST in neural stem cells (NSCs) in mouse hippocampus. These alterations led to a continuous restriction of NSC proliferation toward adulthood, resulting in reduction of neurogenesis associated with the impairment of hippocampal-dependent memory functions. Moreover, we found that voluntary exercise, a widely accepted neurogenic stimulus, restored hippocampal neurogenesis accompanied by the normalization of MDZ-perturbed transcriptome and ameliorated declined cognitive ability in MDZ-exposed mice. Now, we are currently analyzing whether chromatin structural changes are involved in this recovery. Our findings thus pave the way for better understanding of how early-life exposure to anesthesia provokes long-lasting adverse effects on neurocognition and development of a novel therapeutic strategy to prevent delayed-onset learning and memory deficits in people who had experienced anesthesia in their early life. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-165
ネアンデルタール人型GLI3バリアントは多様なヒト表現型に寄与する
Functional contributions of a neanderthal GLI3 variant to various human traits
*阿形 亜子(1)、後藤 仁志(1)、小野 勝彦(1)、野村 真(1)
1. 京都府立医科大学
*Ako Agata(1), Hitoshi Gotoh(1), Katsuhiko Ono(1), Tadashi Nomura(1)
1. Kyoto Prefectural University of Medicine
Keyword: GLI3, evolution, Neanderthal , sonic hedgehog
The evolution of human-specific traits, such as skeletal changes and enlarged brains, is thought to be tightly coupled with changes in genomic structures. Accumulating evidence revealed unique genomic signatures in extinct humans, such as Neanderthal and Denisovans, although phenotypic contributions of lineage-specific genetic variations during human evolution remained elusive. Here, we focused on a Neanderthal variant of GLI3, a transcription factor that is essential for skeletal and brain development. GLI3 acts as transcriptional repressor depending on conserved N-terminal and DNA binding domains, while C-terminus is less conserved among species. All sequenced Neanderthal and Denisovans carry a single amino acid substitution in GLI3 C-terminus (R1537C). We identified that R1537C significantly reduced expression of GLI3 protein, while GLI-dependent transcriptional repression is not affected. RNA-seq analysis confirmed that R1537C altered GLI3-dependent downstream gene profiles, including human specific long non-coding RNAs and sodium-proton exchanger, which are abundantly expressed in the developing and adult central nervous system. Concomitantly, public PheWAS revealed significant associations of GLI3 R1537C with various brain disorders in modern humans. Furthermore, knock-in mice that carry a Neanderthal substitution in Gli3 exhibit altered vertebral morphology. These data suggest that the Neanderthal GLI3 variant affects C-terminus dependent transcriptional regulations, which contributes to the development and evolution of human-specific phenotypes. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-166
オリゴデンドロサイトにおけるRNAヘリカーゼDdx20は神経細胞の生存に関与する
RNA helicase Ddx20 in oligodendrocytes contributes to neuronal survival in the central nervous system
*備前 典久(1)、Anna Simankova(1)、竹林 浩秀(1,2)
1. 新潟大学大学院 医歯学総合研究科 脳機能形態学分野、2. 新潟大学 共用設備基盤センター
*Norihisa Bizen(1), Anna Simankova(1), Hirohide Takebayashi(1,2)
1. Dev Neurobiol Anat, Grad Sch Med Dent, Niigata Univ, Niigata, Japan, 2. CCRF, Niigata Univ, Niigata, Japan
Keyword: Oligodendrocyte, Neurodegeneration, p53, Neuron-glia interaction
Oligodendrocytes contribute to saltatory conduction and axonal protection by forming myelin sheaths surrounding axons. It has been reported that the interaction between oligodendrocyte and neuronal axon is involved in their mutual survival and differentiation; however, the mechanisms have remained unclear. We have previously identified RNA helicase Ddx20 (DEAD box protein 20) as a novel Olig2 binding factor and demonstrated that Ddx20 is indispensable for the development and differentiation of oligodendrocytes (Simankova Glia 2021, Bizen Cell Death Differ 2022). Although mature oligodendrocyte-targeted Ddx20 cKO mice (Mbp-Cre; Ddx20 cKO) show reduced expression of myelin-related genes and hypomyelination between 5 and 6 weeks of age, no apparent abnormalities in oligodendrocyte phenotypes were observed in the early postnatal period. However, the mice revealed activation of the p53 pathway and increased oxidative stress in neurons, leading to neurodegeneration in the hippocampus and thalamus. In addition, microglial activation was found in Ddx20 cKO brains and spinal cords. Interestingly, these phenomena were not observed in jimpy mutant mice, which have gain-of-function (point) mutations in the Plp1 gene and show hypomyelination. Taken together, these results suggest that Ddx20 in oligodendrocytes contributes to neuronal homeostasis through oligodendrocyte-neuron interaction. The activation of the p53 pathway is a common finding in many neurodegenerative diseases, such as Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Therefore, our results suggest a possibility that oligodendrocyte dysfunction may be a common pathogenic factor in many p53-mediated neurodegenerative diseases. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-167
ヒト人工多能性幹細胞の共培養系により形成されたハイブリッドオルガノイドにおける神経分化誘導
Induction of neural differentiation in hybrid organoids formed by co-culture system of human induced pluripotent stem cells
*鈴木 香(1)、池内 真志(2)、林 衆治(1)
1. 一般財団法人 グローバルヘルスケア財団 附属研究所、2. 東京医科歯科大学 生体材料工学研究所 生体機能修復研究部門 バイオデザイン分野
*Kaoru Suzuki(1), Masashi Ikeuchi(2), Shuji Hayashi(1)
1. Res Inst Found Glob Health Care, Nagoya, Japan, 2. Division of Biofunct Restorat Inst Biomaterials Bioengineer Tokyo Med Dent Univ, Tokyo, Japan
Keyword: iPS cell, Neural differentiation, Three-dimension culture, Organoid
Recently, human induced pluripotent stem cell (hiPSC)-derived organoids have been widely used as drug targets for various diseases, and the results have been as expected. It is necessary to develop protocols for the application of early-matured cerebral organoids for drug discovery in neurodegenerative disorders. Organoids can be maintained by inducing vascular differentiation or by long term culturing them in a biodevice with a flow channel (Hofer and Lutolf, Nature Review Materials, 2021). Wörsdörfer et al. demonstrated that the assembly of two types of organoids, those from the mesodermal progenitor cells and hiPSCs, generated vascularized neural organoids (STAR Protocols, 2020). Pasca et al. also demonstrated that assembly oraganoids hiPSCs which were induced different tissues are called “assembloid”, and they showed electrophysiological model from cerebral to peripheral neuronal network (Nat. Biotech, 2020). However, it takes a long period to mature the neural system in these organoids and make them have physiological functions. We attempted several approaches to generate neural activated organoids, which could be matured rapidly to use as a neurodegenerative disease model. One of our approaches involved the production of organoid has vascular circulatory systems using human-derived cells were considered to make hybrid organoids or assembloids. We produced hybrid organoids by co-culturing hiPSCs and human derived cells, each labeled with different wavelength quantum dots and analyzed their dynamics. Additionally, assembloid were also performed, these hybrid organoids and assembloids were differentiated to neural cells and induced cerebral tissue. In this study, we characterized the morphology and gene expression of undifferentiation marker proteins as well as vascular and neural marker proteins in these organoids, and compared them with hiPSC-derived organoids. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-168
転写因子発現で分化誘導されたヒトiPS細胞由来神経細胞における興奮性シナプスの成熟化
Excitatory synapse maturation in transcription factor-induced human iPSC-derived neurons
*林 和花(1)、山田 沙希(1)、渡邊 日佳流(1)、川島 優大(1)、塩本 周作(1)、関野 祐子(2)
1. 株式会社リコー バイオメディカル研究開発室、2. 東京大学大学院薬学系研究科
*Waka Lin(1), Saki Yamada(1), Hikaru Watanabe(1), Yudai Kawashima(1), Shusaku Shiomoto(1), Yuko Sekino(2)
1. Biomed R&D Dept, Ricoh Company, Ltd., Kanagawa, Japan, 2. Grad Sch Ph Sci, Univ of Tokyo, Tokyo, Japan
Keyword: iPS, synapse, neurogenesis, spine
Human induced pluripotent stem cell (iPSC)-derived neurons can be generated by the expression of neurogenic transcription factors and are expected to revolutionize regenerative medicine and disease modeling. However, characterizing the functional maturity of these transcription factor-induced neurons remains a challenge.
To examine the formation of synaptic structures in iPSC-derived neurons, we performed a time-course evaluation of neuronal marker expression and electrophysiological properties for 3 months after induction of differentiation. Transcriptome analysis by RNA sequencing (RNA-seq) confirmed the upregulation of neuronal and synaptic markers. Measurement of electrical activity with a microelectrode array (MEA) system showed spontaneous firing and generation of synchronous network bursts at 1 month of culture. Significant responses to neuromodulators were obtained at later time points, which indicates that multiple neurons connect to each other with functional ion channels and glutamate receptors.
Immunocytochemistry also showed a marked increase of dendrite elongation and formation of presynaptic vesicles after 2 to 3 months of culture. We further evaluated the subcellular localization of drebrin, an actin-binding protein involved in the morphology and dynamics of excitatory dendritic spines. Drebrin clusters were shown to develop along MAP2-positive dendrites after 3 months, which suggests that postsynaptic structures are formed at late culture stages.
Our results show for the first time that transcription factor-induced human iPSC-derived neurons can reach the dendritic spine maturation required for neuroplasticity and response to excitotoxicity. Optimizing culture conditions for synapse maturation would prove useful for the future development of in vitro models that are physiologically relevant to higher brain functions and cognitive disorders. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-169
高速分化技術を用いたヒトiPSC細胞由来アストロサイトとヒト胎児脳由来アストロサイトの機能性比較
Functional comparison between human fetal astrocytes and human iPS cell-derived astrocytes produced by rapid differentiation technology.
*加賀 悠樹(1)、鮫島 達也(1)、腰塚 慎之介(1)、瀬尾 学(2)、荒谷 知行(1)、東 基記(1)
1. 株式会社リコー
*Yuki Kaga(1), Tatsuya Sameshima(1), Shinnosuke Koshizuka(1), Manabu Seo(2), Tomoyuki Aratani(1), Motoki Azuma(1)
1. RICOH Co., Ltd., 2. Elixirgen Scientific, Inc.
Keyword: iPSC, astrocyte, neurons, in vitro assay
Astrocytes constitute a significant proportion of the central nervous system (CNS) cell population and have multiple function that include CNS homeostasis, synapse formation and metabolic support in the CNS. Since it has also been known that astrocyte disfunction causes many neurological disorders, neuron-astrocyte co-culture in vitro system is often used for CNS drug discovery research. Researchers in this field sometimes use commercially available human primary astrocytes. However, since many of them were harvested from human fetal brains, the use of the astrocytes has recently become controversial from an ethical aspect. In addition, although in vivo and in vitro animal models have also been used in CNS drug discovery research, it is known that the models are often insufficient to predict the drug efficacies and toxicities in human. To overcome these limitations, it is expected to develop in vitro evaluation systems with neuron-astrocyte co-cultures differentiated from human-derived induced pluripotent stem cells (hiPSCs) to predict drug efficacies and toxicities in human brain. In this study, we compared hiPSC-derived astrocyte differentiated by Quick-TissueTM technology, which enables hiPSCs to differentiate into target cell types, to human fetal astrocyte, in neuron-astrocyte co-cultures. In this poster, we will show the results of gene expression analysis and pharmacological response of both co-cultures and discuss the functional utility of hiPSC-derived astrocytes in the field of CNS drug discovery. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-170
Development of an In Vitro Platform to Control iPSC Differentiation by Morphogen Gradient
*Koh Isabel(1)、Hagiwara Masaya(1)
*Isabel Siew Yin Koh(1), Masaya Hagiwara(1)
1. Cluster for Pioneering Research, RIKEN, Wako, Japan
Keyword: Morphogen Gradient, Localisation, Organoid, Cube Device
Stem cell and organoid technologies have contributed greatly as in vitro models to expand our understanding of early development. Yet, further progress is limited by the simplicity of current organoid systems. Although cells in vivo are guided by morphogen gradients to develop into tissues with sophisticated complex structures and functions, most organoids are cultured in rather static conditions; cells cultured in a homogeneous medium are exposed to the same differentiation cues from all directions. Microfluidics, on the other hand, enable the establishment of concentration gradients, but usually involve long set-up times and the samples are not easily retrievable from the fluidic chip.
We had previously developed a cube culture device made up of a polycarbonate frame with agarose walls, in which tissue samples can be cultured in an ECM hydrogel. The device allows 3D-cultured samples to be handled with a pair tweezers, which facilitates the quick transfer of samples to a fluidic device. Making use of this device, the aim of this research is to develop a platform in which cells cultured in the cube device can be seamlessly integrated with fluidics to introduce morphogen gradients that direct organoid formation.
To demonstrate this application, human iPSC spheroids were suspended in Matrigel in the cube, then placed in a fluidic chip with two separate chambers for two different media on opposing sides of the cube. One chamber was filled with ectoderm differentiation medium, and the other contained endoderm differentiation medium. After four days of culture, morphological differences between the two sides of the organoids became apparent. When immuno-stained for neural ectoderm marker (βIII-tubulin) and endoderm marker (FoxA2), localisation of the markers at the opposite ends of the organoid was observed. In this way, we could control the differentiation pattern of organoids by generating a gradient of growth factors across the organoid.
The advantage of this platform is that cells cultured in the modular cube can be easily fitted into the fluidic device at the appropriate timing to begin generating a morphogen gradient, and just as easily removed at the end of experiments for further experimental processes. Using this platform, more complex organoids such as organoids with dorsal-ventral or anterior-posterior patterning may be achievable in the future, which can aid in furthering our understanding in human developmental biology by using in vitro models. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-171
胚性幹細胞由来オルガノイド複合体を用いたin vitro非ヒト霊長類脳モデルの構築
An in vitro non-human primate brain model using assembloids derived from embryonic stem cells
*小寺 知輝(1)、原 大樹(1)、森本 菜央(1)、竹内 遼介(1)、小坂田 文隆(1,2,3,4)
1. 名古屋大学大学院創薬科学研究科、2. 名古屋大学高等研究院、3. 名古屋大学未来社会創造機構ナノライフシステム研究所、4. 名古屋大学糖鎖生命コア研究所
*Tomoki Kodera(1), Taiki Hara(1), Nao Morimoto(1), Ryosuke Takeuchi(1), Fumitaka Osakada(1,2,3,4)
1. Grad Sch Pharmaceutical Sci, Nagoya univ, Aichi, Japan, 2. Inst for Adv Res, Nagoya univ, Aichi, Japan, 3. Inst of Nano-Life-Systems, Inst of Innovation for Future Society, Nagoya univ, Aichi, Japan, 4. Inst for Glyco-core Res, Nagoya univ, Aichi, Japan
Keyword: ES cells, organoid, assembloid, marmoset
In drug development, the success rate of new drugs at clinical trials in psychiatric disorders is less than 10%. This low success rate could be attributed to differences in the brain structure and function between rodents and humans. To improve this issue, we should use both in vivo and in vitro models of not only rodents but also non-human primates to evaluate drug effects. We focused on the common marmoset (Callithrix jacchus) as a model animal for studying psychiatric disorders since marmosets have higher-order brain regions such as the prefrontal cortex. Although marmosets are a promising animal model for psychiatric disorders, in vitro models of marmosets are remain lacking.
Three-dimensional cell aggregates, called brain organoids, have enormous potentials for studying brain functions in vitro. Accumulating evidence indicates that brain organoids recapitulate many aspects of brain development and disease. Therefore, constructing an in vitro brain organoid system of non-human primates will facilitate understanding neurobiology and disease mechanisms and assessing pharmacological effects. The present study aims to establish an in vitro brain model of marmosets by inducing cortical and ganglionic eminence (GE) organoids containing both excitatory and inhibitory neurons from marmoset embryonic stem cells (cjESCs).
To generate cortical organoids and GE organoids from cjESCs, we optimized the SFEBq method. We induced organoids positive for the dorsal telencephalon markers Emx2 and Foxg1. We also induced organoids positive for the ventral telencephalon markers Nkx2.1 and Lhx6 by adding SAG, an agonist of Sonic hedgehog (Shh) signaling, in the SFEBq method. Immunohistochemistry showed that the cortical organoids expressed the deep layer neuron markers Tbr1 and Ctip2, whereas the GE organoids expressed inhibitory neuron markers Gad67, PV, and SST. In addition, two-photon microscopy with the genetically encoded calcium sensor GCaMP6 revealed Ca responses in the organoids, suggesting that the induced organoids produced functionally mature neurons. We next fused these two types of organoids in vitro to recapitulate more complex events such as areal interactions.
These results demonstrate that cjESC-derived cortical and GE organoids contain neuronal subtypes specific to their corresponding regions in vivo. Co-culture of these two organoids containing both excitatory and inhibitory neurons will be a versatile in vitro brain model of non-human primates. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-172
ヒトiPS細胞からのサブタイプ特異的な神経細胞への分化誘導法を基盤とした疾患解析プラットフォーム
A platform for disease analysis based on a method for differentiation of human iPS cells into subtype-specific neurons
*石川 充(1)、銭 映美(1)、岡野 栄之(1)
1. 慶應義塾大学
*Mitsuru Ishikawa(1), Emi Qian(1), Hideyuki Okano(1)
1. Keio University
Keyword: iPS cells, Transcription factor, Alzheimer's disease, ALS
To use human induced pluripotent stem cells (iPSCs) and methods to induce neural differentiation from iPSCs have already become a powerful tool for reproduction of neurochemical and neurophysiological functions in vitro, such as the generation of brain organoids. In particular, recent studies using disease-specific iPSC cells have made it possible to obtain more scientifically robust results by using a larger number of samples, including those from patients with sporadic diseases. In addition to this, it is becoming increasingly important to optimize the system for rapid pathological assessment and drug screening.
However, brain organoids have been difficult to analyze due to the need for long-term culture, higher cellular heterogeneity, and higher clonal diversity. Therefore, we turned to the method of inducing neurons in a target-specific manner by transiently expressing transcription factors such as bHLH-type proneural factors in iPSCs. We are modifying this tool with the TetO-NEUROG2 or TetO-ASCL1 systems to develop a platform for stable generation and functional analysis of subtype-specific neurons. We will introduce this new platform and report the results of the studies on neurodegenerative diseases, psychiatric disorders, and neurodevelopmental disorders.
We believe that this platform will enable the selective and robust generation of individual subtype-specific neurons and provide a rapid pathophysiological or drug screening system. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-173
公共データベースを用いた血管化ヒト大脳皮質オルガノイドにおけるシングルセルRNA解析
Single-cell RNA analysis in vascularized human cortical organoids utilizing a public database
*佐藤 由弥(1)、朝日 透(1,2,3)、片岡 孝介(3)
1. 早稲田大学大学院先進理工学研究科、2. 早稲田大学ナノ・ライフ創新研究機構、3. 早稲田大学総合研究機構
*Yuya Sato(1), Toru Asahi(1,2,3), Kosuke Kataoka(3)
1. Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan, 2. Research Organization for Nano & Life Innovation, Waseda University, Tokyo, Japan, 3. Comprehensive Research Organization, Waseda University, Tokyo, Japan
Keyword: ORGANOIDS, SINGLE-CELL, VASCULARIZATION, META-ANALYSIS
ヒト大脳オルガノイド（hCO）は、ヒトの大脳皮質を構造的・機能的に再現するため、ヒト大脳の初期発生や疾患モデルを研究する有用なツールである。しかし、hCOは血管を持たないため、十分な酸素と栄養素が深部まで供給されず、再現性や生存率が低いことが問題となっている。そこで近年、ヒト臍帯静脈内皮細胞（HUVEC）との共培養や、血管内皮細胞の分化に重要である転写因子ETV2の過剰発現など、複数の血管形成手法が提案されている。しかし、これら複数の手法で作製された血管化hCOは、ヒト胎児脳と統合的に評価されていない。そこで本研究では、最適な血管形成手法を探索するために、複数の手法で作製された血管化hCOおよびヒト胎児脳におけるシングルセルRNA-seq（scRNA-seq）データを統合的に比較解析した。そのために、収集した複数のscRNA-seqデータから低品質細胞やマルチプレットなどを前処理により除去し、26,201個の遺伝子発現データを含む130,398細胞からなるデータセットを得た。得られた全ての細胞における遺伝子発現プロファイルをUMAP解析により可視化し、マーカー遺伝子の発現量によって15種類の細胞種を割り当てた。この細胞種は、神経細胞、グリア細胞、血管を構成する壁細胞（ACTA2+／TAGLN+／RGS5+）および内皮細胞（CLDN5+／PECAM1+）に類似した細胞、繊毛を持つ上衣細胞に類似した細胞（FOXJ1+／NPHP1+／MNS1+）を含んでいた。さらに、Gene Set Enrichment Analysis（GSEA）解析によって血管化に伴い発現変動した遺伝子群の機能を調べた。その結果、HUVECとの共培養による血管化に伴って、リボソーム生合成やタンパク質翻訳に関わる遺伝子群の発現量の上昇や、低酸素応答遺伝子群の発現量の減少が認められた。本結果は、HUVECとの共培養によって細胞への栄養供給が増加し、低酸素ストレスが低減することを示唆している。同様に、ETV2の過剰発現による血管化に伴う発現変動遺伝子群をGSEA解析によって調べた結果、血管化に伴って筋肉や細胞外マトリックスに関わる遺伝子群の発現量の減少が認められた。ETV2の過剰発現による血管化の機構について精査するため、推定分化経路における遺伝子の発現変動を解析した。その結果、ETV2の過剰発現により、内皮細胞において血管形成に重要な役割を果たすVEGF受容体（FLT1およびHAND1）の遺伝子の発現量が、特定の時期において上昇していることが明らかになった。この結果は、ETV2の過剰発現によってVEGF受容体が限られた時期に活性化することで、血管化が誘導されることを示唆している。以上より、異なる血管形成手法の単一細胞レベルでの分子生物学的特徴の一端が明らかになった。これらの結果は、hCOにおける血管化やヒト脳における血管形成機構に新たな知見を提供すると期待される。 | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-174
成体マウス脳において交連下器官は神経上皮細胞の特徴を維持する
The subcommissural organ maintains features of neuroepithelial cells in the adult mouse
*稲田 仁(1,2)、Laarni Grace Corales(2)、平岡 宏太良(3)、大隅 典子(2)
1. 東北大学大学院医工学研究科、2. 東北大学大学院医学系研究科、3. 東北大学サイクロトロン・RIセンター
*Hitoshi Inada(1,2), Laarni Grace Corales(2), Kotaro Hiraoka(3), Noriko Osumi(2)
1. Grad Sch Biomed Eng, Tohoku Univ, Japan, 2. Grad Sch Med, Tohoku Univ, Japan, 3. Cyclo and Radio Center, Tohoku Univ, Japan
Keyword: subcommissural organ , Pax6, Sox2, PCNA
The subcommissural organ (SCO) is a part of the circumventricular organs located in the dorsocaudal region of the third ventricle at the entrance of the aqueduct of Sylvius. The SCO comprises epithelial cells and produces high molecular weight glycoproteins, which are secreted into the third ventricle and become part of Reissner’s fiber in the cerebrospinal fluid. Abnormal development of the SCO has been linked with congenital hydrocephalus, a condition characterized by excessive accumulation of cerebrospinal fluid in the brain. In the present study, we characterized the SCO cells in the adult mouse brain to gain insights into the possible role of this brain region. Immunohistochemical analyses revealed that expression of Pax6, a transcription factor essential for SCO differentiation during embryogenesis, is maintained in the SCO at postnatal stages from P0 to P84. SCO cells in the adult brain expressed known neural stem/progenitor cell (NSPC) markers, Sox2, and vimentin. The adult SCO cells also expressed proliferating marker PCNA, although another proliferation marker, Ki67, indicating a G2/M phase, was not detected. The SCO cells did not incorporate BrdU, a marker for DNA synthesis in the S phase. Therefore, the SCO cells have a potential for proliferation but are quiescent for cell division in the adult mouse brain. The SCO cells also expressed GFAP, a marker for astrocytes or NSPCs, but not other differentiation markers such as NeuN (for neurons) nor Olig2 (for oligodendrocytes). A few cells positive for Iba1 (microglia) and PDGFRα (oligodendrocyte progenitors) existed within or on the periphery of the SCO, respectively. These findings revealed that the SCO cells have a unique feature as secretory yet immature neuroepithelial cells in the adult mouse brain. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-175
異種感覚の可塑性におけるアストロサイトの変化
The alteration of astrocytes in cross-modal plasticity
*竹田 育子(1,2)
1. 名古屋大学大学院医学系研究科分子細胞学、2. 生理学研究所多細胞回路動態研究部門
*Ikuko Takeda(1,2)
1. Department of Anatomy and Molecular Cell Biology, Grad Sch Med, Nagoya university, Nagoya, Japan, 2. Division of Multicellular Circuit Dynamics, National Institute for Physiological Sciences
Keyword: cross-modal plasticity, astrocyte
Cross-modal plasticity is an adaptive process that compensates for sensory loss. Even though the loss of sensory input to a cortical region promotes functional and structural plastic changes in cross-modal plasticity, the role of astrocyte that can mediate neural plasticity is unclear. In this study, we investigated the role of astrocytes in cross-modal plasticity, specifically whether they are relevant to residual sensory function improvement. We studied inter-sensory connection in mouse primary sensory cortex barrel field (S1BF) and secondary visual cortex (V2L) following monocular deprivation (MD). Our recent studies report that MD in mice promotes increased functional connectivity from S1BF to V2L. We performed MD by right eye either before (2-week) or after (5-week) eye opening to assess morphological and functional changes of astrocytes in S1BF and V2L. Numbers of S1BF neurons and V2L astrocytes were increased in 5-week old MD mice but not in 2-week old MD mice. The number of S1BF/V2L astrocytes contacting their somas to neighboring neuronal somas is also increased in 5-week old MD mice but not in 2-week old MD mice, suggesting V2L neurons which projected from other primary sensory legion induce proliferation of astrocytes that may cause reciprocal activity modulation with contacted neurons in 5-week old MD. This correlated with upregulated astrocytic DNA proliferation, as indicated by increased numbers of S1BF/V2L EdU positive astrocytes, in 5-week old MD mice. To study how astrocyte response with neuronal signaling especially with inter sensory connection, we visualized astrocyte Ca2+ activity in response to whisker stimulation in vivo using two-photon microscope. In chronic phase of MD, V2L astrocytes showed higher intensity of Ca2+ activities relative to control mice, in 5-week old MD mice. Whereas in 2-week old MD mice, V2L astrocyte Ca2+ activities were comparable to control mice. Alterations in inter sensory connectivity may increase the activity of some astrocytes around neurons that receive input from S1BF only in 5-week old MD mice to induce astrocytic proliferation . These results suggest that cross-modal plasticity proceeds via differing mechanisms that are specific to the age of vision-loss. We expect that clarifying the mechanisms underpinning cross-modal plasticity will be important for developing an effective approach for driving the construction of circuits which benefit the restoration of sensory deficits. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-177
分泌性糖タンパク質リーリンによるN-カドヘリン輸送制御
Regulation of N-cadherin trafficking by a secretory glycoprotein Reelin
*林 周宏(1)、久保 健一郎(1,2)、仲尾 信彦(3,4)、安達 泰治(3,4)、仲嶋 一範(1)
1. 慶應義塾大学 医学部、2. 東京慈恵会医科大学、3. 京都大学 大学院工学研究科、4. 京都大学 ウイルス・再生医科学研究所
*Kanehiro Hayashi(1), Ken-ichiro Kubo(1,2), Nobuhiko Nakao(3,4), Taiji Adachi(3,4), Kazunori Nakajima(1)
1. Keio University School of Medicine, 2. The Jikei University School of Medicine, 3. Graduate School of Engineering, Kyoto University, 4. Institute for Frontier Life and Medical Sciences, Kyoto University
Keyword: cortical development, Reelin, N-cadherin
In the mammalian neocortex, excitatory neurons that are born around the same time are aligned in parallel with the brain surface, forming a layered structure. In addition, early-born and late-born neurons are located in the deep and superficial region in the cortex, respectively, resulting in the birthdate-dependent inside-out pattern of layer formation. The secretory protein Reelin is a glycoprotein expressed in Cajal-Retzius cells in the marginal zone of the cerebral cortex and affects migrating neurons. The Reelin deficient mouse reeler exhibits disordered layer structure in an almost outside-in pattern, indicating the importance of Reelin in the cortical layer formation. However, how Reelin controls neuronal positioning is not fully understood. We previously reported that ectopic expression of Reelin in the mouse developing neocortex caused formation of a characteristic neuronal aggregate with a dendrite-rich center and a cell body-rich periphery, resembling the structure of the marginal zone and the most superficial region of the cortical plate, respectively (Kubo et al., 2010; Sekine et al., 2011, 2012; Hirota et al., 2018, 2020). Furthermore, we also reported N-cadherin is involved in the formation of the aggregates (Matsunaga, et al., 2017).
In the present study, we report that intracellular trafficking of N-cadherin is controlled by Reelin. Treatment of primary cultured cortical neurons with Reelin increased the protein amount and the adhesion strength of N-cadherin on the cellular surface. Migrating neurons in their final stage extend their leading processes into the marginal zone where Reelin is abundant, and the experiment using in utero electroporation and CRISPR-Cas9 knock-in system showed N-cadherin was localized in these processes. These results suggest that Reelin promotes the cell adhesion on migrating neurons by transferring N-cadherin to neuronal processes. | | 2022年7月1日 11:00～12:00 宜野湾市民体育館 ポスター会場2
2P-178
アカハライモリ (Cynops pyrrhogaster)の再生におけるIGFBP (insulin-growth factor binding protein)ドメインの新規役割の可能性
Potential role of IGFBP (insulin-growth factor binding protein) domain in japanese newt (Cynops pyrrhogaster) regeneration
*若松 華蓮(1,2)、衣笠 ジョシュア(1)、辻 敏之(2)、Pavel Prosselkov(1)
1. Manai Institute of Science and Technology、2. 三田国際学園高等学校
*Karen Wakamatsu(1,2), Joshua Kai Karpelowitz(1), Toshiyuki Tsuji(2), Pavel Prosselkov(1)
1. Manai Institute of Science and Technology, 2. Mita International School
Keyword: nsCCN, IGFBP, Regeneration
Among many existing animal models, an exceptional ability of newts to restore a lost limb has been given a substantial attention. Lesion of a body part triggers an expression of a number of genes at the injury site gradually initiating the regeneration process. At present, the newt regeneration process has become more or less elucidated, however, the whole picture of the genes involved is still unknown, and the processes occurring in the cell have not been fully modelled.
We have previously reported the presence of IGFBP domain containing RNA transcripts in the regenerating newt limb RNA-seq (1). Protein predictions have robustly pointed to a potential translatability of the IGFBP along with other downstream domains. The exclusive presence of IGFBP in the regenerating newt limb and its absence in a control non-damaged tissue is enigmatic and thrilling, pointing to a novel mechanism underlying a path to the damaged tissue regeneration via a blastema formation. In order to explore further the functionality of IGFBP domain, we are currently analyzing the RNA expression profiles in the eye, retina and embryo of newts using publicly available RNA-seq data sources and planning to perform in situ hybridization to validate the localization and time frames of IGFBP expression during the blastema formation and subsequent limb regeneration. We were successful in validating the presence of the encoded IGFBP within the novel newt specific CCN family related gene (nsCCN) by sequencing and have generated the in situ probes.
(1) Wakamatsu K, Karpelowitz, J, Tsuji T, Prosselkov P. (2021) "Transcriptome characterization of a novel newt specific CCN family related gene involved in regeneration" 44th JNS-2021, Kobe, 28th-31st Jul | | | |
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